http://2010.igem.org/wiki/index.php?title=Special:Contributions&feed=atom&limit=250&target=Lior&year=&month=2010.igem.org - User contributions [en]2024-03-28T22:27:07ZFrom 2010.igem.orgMediaWiki 1.16.5http://2010.igem.org/Team:Debrecen-Hungary/video_projectTeam:Debrecen-Hungary/video project2010-10-27T18:07:40Z<p>Lior: /* Links */</p>
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="Film In Lab" - The Video Tutorial Project=<br />
<br />
<br />
<br />
<br />
The iGEM – Debrecen group is a multidisciplinary team. In summer 2010 we had guest students, the AKG team, from a high school in Budapest joining us. Our aim was to bring all students, coming from different backgrounds, into a functioning unit. Thus, we have created as part of the preparatory phase the video tutorials. The video tutorials project, also called:"Film in Lab" project, started as an initiative of our group and grew larger.<br />
<br />
<br />
<br />
In general, the idea was to create an observational way of teaching the basic lab techniques. Nevertheless, the project got lots of empathy, thus we have decided to improve it by adding soundtracks, pearls and comments. For this, we have created an own sub team that produced and created the "Film in Lab" project. This way we are aiming to establish an open source that will serve future iGEM members around the globe. We believe that this contribution is in harmony with the idea of the iGEM initiative. <br />
<br />
<br />
<br />
Moreover, this "Film in Lab" project is a great example for how complicated procedures can be simplified; and resulting in efficient application of the theoretical knowledge by a beginner.<br />
For making the experience complete and fruitful, one may find the protocols in the [http://www.openwetware.org/wiki/Special:Contributions/Yakir_Guri 'Open Wet Ware'] account of iGEM-Debrecen Team 2010. We recommend to read the protocols carefully before and after watching the video tutorial for maximizing the experience.<br />
<br />
<br />
<br />
We thank'' 'Roche''' for supporting and sponsoring this project. Also, we thank all students members and supervisors for critical review of scripts and audio soundtracks. Especially, to Dorottya Jakab (AKG team) and Burger Veronika (a sixth year medical student) who made this project posible.<br />
<br />
<br />
See more at our [http://www.youtube.com/user/debrecenigem2010 Youtube channel]. <br><br />
Download our video project summary with links to the protocols [https://static.igem.org/mediawiki/2010/8/8f/Links_to_OWW_and_YOUTUBE.pdf here].<br />
<br />
<br />
<h5>The members of the "Film in Lab" project :</h5><br />
<br />
<br />
Dorottya, Jakab – presenting student (Transformation, Midiprep, Gel-Electrophoresis)<br />
<br />
Endre, Kristof – video making (PCR)<br />
<br />
Guri, Yakir – supervising the project, film making, editing protocols and videos<br />
<br />
Liu, Shun-Chien – coordinator, Scripts, video making (nanodrop) and audio co-editor <br />
<br />
Malka, Lior – presenting student (nanodrop),and web master<br />
<br />
Markovits, Daniel – vocals <br />
<br />
Nagy, Kata - critical review of Midiprep<br />
<br />
Ozgyin, Lilla – presenting student (Cell-passaging, Ligand stimulation, Transfection)<br />
<br />
Sandor, Kata – presenting student (PCR)<br />
<br />
<br><br><br />
<br />
''Guri, Yakir''<Br><br />
<br />
Supervisor of the video project <br />
<br />
<br />
<br />
''Balint L. Balint'', MD, PhD,<br />
<br />
Supervisor and instructor of 'iGEM-Debrecen 2010 Team' <br />
<br><br><br><br />
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=References=<br />
[https://2010.igem.org/Team:Debrecen-Hungary/protocols/makinglb ''Main article - Making Lurea's Broth'']<br />
<br />
[http://www.openwetware.org/wiki/IGEM:University_of_Debrecen:Transformation_of_competent_cells ''Main article - Transformation of competent cells'']<br />
<br />
[http://openwetware.org/wiki/Editing_IGEM:University_of_Debrecen:_Mini_Prep ''Main article - Mini prep'']<br />
<br />
[http://openwetware.org/wiki/IGEM:University_of_Debrecen:Restriction_biobrick_parts#Notes ''Main article - Restriction enzyme digestion'']<br />
<br />
[http://www.openwetware.org/wiki/IGEM:University_of_Debrecen:PCR_purification_from_Agarose_Gel ''Main article - PCR product purification / Gel purification''] <br><br />
<br />
[http://www.openwetware.org/wiki/IGEM:University_of_Debrecen:_Polymerase_Chain_Reaction_%28PCR%29 ''Main article - PCR''] <br><br />
<br />
[http://openwetware.org/wiki/IGEM:University_of_Debrecen:Gel_electrophoresis ''Main article - Gel electrophoresis'']<br />
<br />
[http://www.openwetware.org/wiki/IGEM:University_of_Debrecen:Cell_Passaging ''Main article - Cell passaging'']<br />
<br />
[http://openwetware.org/wiki/IGEM:University_of_Debrecen:_Media_PEI_Preparation ''Main article - Media PEI preparation'']<br />
<br />
[http://openwetware.org/wiki/IGEM:University_of_Debrecen:_transfection ''Main article - Transfection'']<br />
<br />
[http://openwetware.org/wiki/IGEM:University_of_Debrecen:Ligand_Treatment ''Main article - Ligand treatment''] <br />
<br />
[http://openwetware.org/wiki/IGEM:University_of_Debrecen:_Enzymatic_activity_with_Victor_machine ''Main article - Measuring Luciferase activity with the Victor plate reader'']<br />
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</html></div>Liorhttp://2010.igem.org/Team:Debrecen-Hungary/video_projectTeam:Debrecen-Hungary/video project2010-10-27T18:06:54Z<p>Lior: /* "Film In Lab" - The Video Tutorial Project */</p>
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<br />
<div align="justify"><br />
<br />
="Film In Lab" - The Video Tutorial Project=<br />
<br />
<br />
<br />
<br />
The iGEM – Debrecen group is a multidisciplinary team. In summer 2010 we had guest students, the AKG team, from a high school in Budapest joining us. Our aim was to bring all students, coming from different backgrounds, into a functioning unit. Thus, we have created as part of the preparatory phase the video tutorials. The video tutorials project, also called:"Film in Lab" project, started as an initiative of our group and grew larger.<br />
<br />
<br />
<br />
In general, the idea was to create an observational way of teaching the basic lab techniques. Nevertheless, the project got lots of empathy, thus we have decided to improve it by adding soundtracks, pearls and comments. For this, we have created an own sub team that produced and created the "Film in Lab" project. This way we are aiming to establish an open source that will serve future iGEM members around the globe. We believe that this contribution is in harmony with the idea of the iGEM initiative. <br />
<br />
<br />
<br />
Moreover, this "Film in Lab" project is a great example for how complicated procedures can be simplified; and resulting in efficient application of the theoretical knowledge by a beginner.<br />
For making the experience complete and fruitful, one may find the protocols in the [http://www.openwetware.org/wiki/Special:Contributions/Yakir_Guri 'Open Wet Ware'] account of iGEM-Debrecen Team 2010. We recommend to read the protocols carefully before and after watching the video tutorial for maximizing the experience.<br />
<br />
<br />
<br />
We thank'' 'Roche''' for supporting and sponsoring this project. Also, we thank all students members and supervisors for critical review of scripts and audio soundtracks. Especially, to Dorottya Jakab (AKG team) and Burger Veronika (a sixth year medical student) who made this project posible.<br />
<br />
<br />
See more at our [http://www.youtube.com/user/debrecenigem2010 Youtube channel]. <br><br />
Download our video project summary with links to the protocols [https://static.igem.org/mediawiki/2010/8/8f/Links_to_OWW_and_YOUTUBE.pdf here].<br />
<br />
<br />
<h5>The members of the "Film in Lab" project :</h5><br />
<br />
<br />
Dorottya, Jakab – presenting student (Transformation, Midiprep, Gel-Electrophoresis)<br />
<br />
Endre, Kristof – video making (PCR)<br />
<br />
Guri, Yakir – supervising the project, film making, editing protocols and videos<br />
<br />
Liu, Shun-Chien – coordinator, Scripts, video making (nanodrop) and audio co-editor <br />
<br />
Malka, Lior – presenting student (nanodrop),and web master<br />
<br />
Markovits, Daniel – vocals <br />
<br />
Nagy, Kata - critical review of Midiprep<br />
<br />
Ozgyin, Lilla – presenting student (Cell-passaging, Ligand stimulation, Transfection)<br />
<br />
Sandor, Kata – presenting student (PCR)<br />
<br />
<br><br><br />
<br />
''Guri, Yakir''<Br><br />
<br />
Supervisor of the video project <br />
<br />
<br />
<br />
''Balint L. Balint'', MD, PhD,<br />
<br />
Supervisor and instructor of 'iGEM-Debrecen 2010 Team' <br />
<br><br><br><br />
<br />
<html><br />
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<img src="https://static.igem.org/mediawiki/2010/b/be/Roche.jpg<br />
"></a><br />
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<br />
=Links=<br />
[https://2010.igem.org/Team:Debrecen-Hungary/protocols/makinglb ''Main article - Making Lurea's Broth'']<br />
<br />
[http://www.openwetware.org/wiki/IGEM:University_of_Debrecen:Transformation_of_competent_cells ''Main article - Transformation of competent cells'']<br />
<br />
[http://openwetware.org/wiki/Editing_IGEM:University_of_Debrecen:_Mini_Prep ''Main article - Mini prep'']<br />
<br />
[http://openwetware.org/wiki/IGEM:University_of_Debrecen:Restriction_biobrick_parts#Notes ''Main article - Restriction enzyme digestion'']<br />
<br />
[http://www.openwetware.org/wiki/IGEM:University_of_Debrecen:PCR_purification_from_Agarose_Gel ''Main article - PCR product purification / Gel purification''] <br><br />
<br />
[http://www.openwetware.org/wiki/IGEM:University_of_Debrecen:_Polymerase_Chain_Reaction_%28PCR%29 ''Main article - PCR''] <br><br />
<br />
[http://openwetware.org/wiki/IGEM:University_of_Debrecen:Gel_electrophoresis ''Main article - Gel electrophoresis'']<br />
<br />
[http://www.openwetware.org/wiki/IGEM:University_of_Debrecen:Cell_Passaging ''Main article - Cell passaging'']<br />
<br />
[http://openwetware.org/wiki/IGEM:University_of_Debrecen:_Media_PEI_Preparation ''Main article - Media PEI preparation'']<br />
<br />
[http://openwetware.org/wiki/IGEM:University_of_Debrecen:_transfection ''Main article - Transfection'']<br />
<br />
[http://openwetware.org/wiki/IGEM:University_of_Debrecen:Ligand_Treatment ''Main article - Ligand treatment''] <br />
<br />
[http://openwetware.org/wiki/IGEM:University_of_Debrecen:_Enzymatic_activity_with_Victor_machine ''Main article - Measuring Luciferase activity with the Victor plate reader'']<br />
</div><br />
<br />
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</html></div>Liorhttp://2010.igem.org/Team:Debrecen-Hungary/galleryTeam:Debrecen-Hungary/gallery2010-10-27T17:52:46Z<p>Lior: </p>
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<li><a href="https://static.igem.org/mediawiki/2010/d/d3/JCF5303.JPG"><img src="https://static.igem.org/mediawiki/2010/d/d3/JCF5303.JPG" alt="" title="" /></a></li><br />
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__NOTOC__<br />
<div align="justify"><br />
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</div></div>Liorhttp://2010.igem.org/File:JCF5303.JPGFile:JCF5303.JPG2010-10-27T17:51:52Z<p>Lior: </p>
<hr />
<div></div>Liorhttp://2010.igem.org/File:DSC02202.JPGFile:DSC02202.JPG2010-10-27T17:45:17Z<p>Lior: </p>
<hr />
<div></div>Liorhttp://2010.igem.org/File:DSC02205.JPGFile:DSC02205.JPG2010-10-27T17:42:25Z<p>Lior: </p>
<hr />
<div></div>Liorhttp://2010.igem.org/Team:Debrecen-Hungary/galleryTeam:Debrecen-Hungary/gallery2010-10-27T17:40:36Z<p>Lior: </p>
<hr />
<div>{{Template:Debrecen-Hungary_secret}}<br />
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The original version of this stylesheet and the associated (x)html<br />
is available at http://www.cssplay.co.uk/menu/lightbox.html<br />
Copyright (c) 2005-2007 Stu Nicholls. All rights reserved.<br />
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a {color:#ede8e2;}<br />
a:hover {text-decoration:none;}<br />
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{display:none;}<br />
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<li><a href="https://static.igem.org/mediawiki/2010/2/21/0006.JPG"><img src="https://static.igem.org/mediawiki/2010/2/21/0006.JPG" alt="" title="" /></a></li><br />
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<li><a href="https://static.igem.org/mediawiki/2010/0/04/00017.jpg"><img src="https://static.igem.org/mediawiki/2010/0/04/00017.jpg" alt="" title="" /></a></li><br />
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<li><a href="https://static.igem.org/mediawiki/2010/a/a9/Pi3.jpg"><img src="https://static.igem.org/mediawiki/2010/a/a9/Pi3.jpg" alt="" title="" /></a></li><br />
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<li><a href="https://static.igem.org/mediawiki/2010/1/11/Image044.jpg"><img src="https://static.igem.org/mediawiki/2010/1/11/Image044.jpg" alt="" title="" /></a></li><br />
<br />
<!--- <li><a href="https://static.igem.org/mediawiki/2010/9/91/00026.jpg"><img src="https://static.igem.org/mediawiki/2010/9/91/00026.jpg" alt="" title="" /></a></li> ---><br />
<br />
<br><br><Br><br />
<br />
<br><br><br><br />
<center><br />
<br><br><br><br />
<a href="https://2010.igem.org/Team:Debrecen-Hungary"><img src="https://static.igem.org/mediawiki/2010/9/96/Backbuttonbck.jpg"></a><br />
</center><br />
</ul><br />
<!--[if lte IE 6]></td></tr></table></a><![endif]--><br />
</li><br />
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__NOTOC__<br />
<div align="justify"><br />
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</div></div>Liorhttp://2010.igem.org/File:Image044.jpgFile:Image044.jpg2010-10-27T17:39:51Z<p>Lior: uploaded a new version of "Image:Image044.jpg"</p>
<hr />
<div></div>Liorhttp://2010.igem.org/File:Image044.jpgFile:Image044.jpg2010-10-27T17:38:12Z<p>Lior: </p>
<hr />
<div></div>Liorhttp://2010.igem.org/File:DSC02220.jpgFile:DSC02220.jpg2010-10-27T17:35:36Z<p>Lior: </p>
<hr />
<div></div>Liorhttp://2010.igem.org/Template:Debrecen-Hungary_secretTemplate:Debrecen-Hungary secret2010-10-27T16:32:03Z<p>Lior: </p>
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<body id="template:debrecen-hungary_secret" ></body></html><br />
{|<br />
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|width="1100px" style="padding: 0 0px 0px 0px; background-color:#ede8e2"|<br />
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<style><br />
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<br />
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a:hover breaks li:hover in FF2.<br />
*/<br />
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div.MenuBar ul li:hover ul.DropDownMenu li:hover ul.SideMenu,<br />
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Keep it sync with the drop-down menu width.<br />
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div.MenuBar ul li:hover ul.DropDownMenu li:hover ul.SideMenu li a,<br />
div.MenuBar ul li a:hover ul.DropDownMenu li a:hover ul.SideMenu li a {<br />
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Use MenuTailor.css to customize. */<br />
font: 12px arial, helvetica, sans-serif; /* Required for IE55. */<br />
left: 13em; /* Places the side menu to the right of the drop-down menu.<br />
Keep it sync with the drop-down menu width.<br />
Use MenuTailor.css to customize. */<br />
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text-align: left;<br />
font: 12px arial, helvetica, sans-serif; /* Required for IE55. */<br />
left: 13em; /* Places the side menu to the right of the drop-down menu.<br />
Keep it sync with the drop-down menu width.<br />
Use MenuTailor.css to customize. */<br />
}<br />
/*----------------------------------------------------------------------------- Browser Specific */<br />
* html div.MenuBar ul li a {<br />
float: left; /* Required for IE55 and IE6.<br />
Breaks O9.<br />
Hidden (* html) from non-IE browsers. */<br />
}<br />
* html ul.DropDownMenu li a:hover {<br />
cursor: hand; /* Required for IE55.<br />
Hidden (* html) from non-IE browsers. */<br />
}<br />
ul.DropDownMenu li a:hover {<br />
cursor: pointer; /* Required for IE6 and IE7.<br />
Hidding it (* html) from non-IE browsers breaks IE7! <br />
}<br />
* html div.MenuBar a:hover {<br />
text-decoration: none; /* Required for IE55 and IE6.<br />
Hidden (* html) from non-IE browsers. */<br />
}<br />
* html div.MenuBar ul li table,<br />
* html div.MenuBar ul li table td {<br />
border: 0; /* Required for IE55 and IE6.<br />
Hidden (* html) from non-IE browsers. */<br />
}<br />
/*------------------------------------------------------------------------------- Default Colors */<br />
div.MenuBar {<br />
background-color: Menu;<br />
border-bottom: 1px solid ButtonShadow;<br />
}<br />
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color: MenuText;<br />
}<br />
div.MenuBar ul li:hover a,<br />
div.MenuBar ul li a:hover {<br />
color: #ea7f16;<br />
background-color: Highlight; /* Top-level selected item. */<br />
color: HighlightText;<br />
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/*...............................................................................................*/<br />
div.MenuBar ul li:hover ul.DropDownMenu,<br />
div.MenuBar ul li a:hover ul.DropDownMenu {<br />
background-color: ButtonShadow; /* Sets the drop-down menu "effective border" color. */<br />
}<br />
div.MenuBar ul li:hover ul.DropDownMenu li a,<br />
div.MenuBar ul li a:hover ul.DropDownMenu li a {<br />
background-color: Menu; Drop-down menu unselected items.<br />
Sets the drop-down menu "effective background" color. */<br />
color: MenuText;<br />
}<br />
div.MenuBar ul li:hover ul.DropDownMenu li:hover a,<br />
div.MenuBar ul li a:hover ul.DropDownMenu li a:hover {<br />
background-color: Highlight; /* Drop-down menu selected item. */<br />
color: HighlightText;<br />
}<br />
/*...............................................................................................*/<br />
div.MenuBar ul li:hover ul.DropDownMenu li:hover ul.SideMenu,<br />
div.MenuBar ul li a:hover ul.DropDownMenu li a:hover ul.SideMenu {<br />
background-color: ButtonShadow; /* Sets the side menu "effective border" color. */<br />
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<li><a href="https://2010.igem.org/Team:Debrecen-Hungary/video_project"><span><span>Video Project</span></span></a></li><br />
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</html></div>Liorhttp://2010.igem.org/Team:Debrecen-Hungary/video_projectTeam:Debrecen-Hungary/video project2010-10-27T16:30:54Z<p>Lior: </p>
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<br />
="Film In Lab" - The Video Tutorial Project=<br />
<br />
<br />
<br />
<br />
The iGEM – Debrecen group is a multidisciplinary team. In summer 2010 we had guest students, the AKG team, from a high school in Budapest joining us. Our aim was to bring all students, coming from different backgrounds, into a functioning unit. Thus, we have created as part of the preparatory phase the video tutorials. The video tutorials project, also called:"Film in Lab" project, started as an initiative of our group and grew larger.<br />
<br />
<br />
<br />
In general, the idea was to create an observational way of teaching the basic lab techniques. Nevertheless, the project got lots of empathy, thus we have decided to improve it by adding soundtracks, pearls and comments. For this, we have created an own sub team that produced and created the "Film in Lab" project. This way we are aiming to establish an open source that will serve future iGEM members around the globe. We believe that this contribution is in harmony with the idea of the iGEM initiative. <br />
<br />
<br />
<br />
Moreover, this "Film in Lab" project is a great example for how complicated procedures can be simplified; and resulting in efficient application of the theoretical knowledge by a beginner.<br />
For making the experience complete and fruitful, one may find the protocols in the [http://www.openwetware.org/wiki/Special:Contributions/Yakir_Guri 'Open Wet Ware'] account of iGEM-Debrecen Team 2010. We recommend to read the protocols carefully before and after watching the video tutorial for maximizing the experience.<br />
<br />
<br />
<br />
We thank'' 'Roche''' for supporting and sponsoring this project. Also, we thank all students members and supervisors for critical review of scripts and audio soundtracks. Especially, to Dorottya Jakab (AKG team) and Burger Veronika (a sixth year medical student) who made this project posible.<br />
<br />
<br />
See more at our [http://www.youtube.com/user/debrecenigem2010 Youtube channel]. <br><br />
Download our video project summary with links to the protocols [https://static.igem.org/mediawiki/2010/8/8f/Links_to_OWW_and_YOUTUBE.pdf here].<br />
<br />
<br />
<h5>The members of the "Film in Lab" project :</h5><br />
<br />
<br />
Dorottya, Jakab – presenting student (Transformation, Midiprep, Gel-Electrophoresis)<br />
<br />
Endre, Kristof – video making (PCR)<br />
<br />
Guri, Yakir – supervising the project, film making, editing protocols and videos<br />
<br />
Liu, Shun-Chien – coordinator, Scripts, video making (nanodrop) and audio co-editor <br />
<br />
Malka, Lior – presenting student (nanodrop),and web master<br />
<br />
Markovits, Daniel – vocals <br />
<br />
Nagy, Kata - critical review of Midiprep<br />
<br />
Ozgyin, Lilla – presenting student (Cell-passaging, Ligand stimulation, Transfection)<br />
<br />
Sandor, Kata – presenting student (PCR)<br />
<br />
<br><br><br />
<br />
''Guri, Yakir''<Br><br />
<br />
Supervisor of the video project <br />
<br />
<br />
<br />
''Balint L. Balint'', MD, PhD,<br />
<br />
Supervisor and instructor of 'iGEM-Debrecen 2010 Team' <br />
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</html></div>Liorhttp://2010.igem.org/File:Links_to_OWW_and_YOUTUBE.pdfFile:Links to OWW and YOUTUBE.pdf2010-10-27T16:28:51Z<p>Lior: </p>
<hr />
<div></div>Liorhttp://2010.igem.org/Team:Debrecen-Hungary/protocolsTeam:Debrecen-Hungary/protocols2010-10-27T12:20:37Z<p>Lior: /* Online References At Openwetware */</p>
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=Welcome To Our Protocols=<br />
<br />
<h1> "Science is nothing else but the art of proper reproducibility. " </h1> <br><br />
The iGEM experience is not merely a project or a conference, but it was the way that most of our students got acquainted with the world of biological research laboratory. Pipettes, solutions, gels, electrodes, dishes and other scary machinery quickly filled our lives. <br />
From day one we saw the vast importance of teaching our students to keep a proper laboratory journal. As time passed and our project grew., and with growth sprouted the idea of keeping an electronic laboratory journal with texts and video’s depicting the proper way of doing our niche of science. And so, we present to you our combined effort a text and video version<br />
<br />
=='''Contents'''==<br />
<br />
'''Notebook protocols utilized by the bacterial work subteam '''<br />
<br />
[[Team:Debrecen-Hungary/protocols#Making Lurea's Broth|Making Lurea's Broth]] - [[Team:Debrecen-Hungary/protocols#Transformation of competent cells|Transformation of competent cells]] - [[Team:Debrecen-Hungary/protocols#Mini Prep|Mini Prep]] - [[Team:Debrecen-Hungary/protocols#Midi Prep|Midi Prep]]<br />
<br />
'''Notebook protocols utilized by the molecular tools subteam '''<br />
<br />
[[Team:Debrecen-Hungary/protocols#Restriction enzyme digestion|Restriction enzyme digestion]] - [[Team:Debrecen-Hungary/protocols#Cutting gel for PCR product purification |PCR Purification]] - [[Team:Debrecen-Hungary/protocols#PCR|PCR]] - [[Team:Debrecen-Hungary/protocols#Gel electrophoresis|Gel electrophoresis]] <br />
<br />
'''Notebook protocols utilized by the tissue culture subteam'''<br />
<br />
[[Team:Debrecen-Hungary/protocols#Cell Passaging|Cell Passaging]] - [[Team:Debrecen-Hungary/protocols#Media PEI Preparation |Media PEI Preparation ]] - [[Team:Debrecen-Hungary/protocols#Transfection|Transfection ]] - [[Team:Debrecen-Hungary/protocols#Ligand Treatment |Ligand Treatment ]]<br />
<br />
'''Notebook protocols utilized by the Luciferase subteam'''<br />
<br />
[[Team:Debrecen-Hungary/protocols#Measuring Luciferase activity with the Victor plate reader |Measuring Luciferase activity with the Victor plate reader ]]<br />
<br />
== Notebook protocols utilized by the bacterial work subteam ==<br />
<br />
=== Making Lurea's Broth===<br />
<br />
[https://2010.igem.org/Team:Debrecen-Hungary/protocols/makinglb Read more...]<br />
<br />
[[Team:Debrecen-Hungary/protocols#Notebook|[TOP]]]<br />
<br />
=== Transformation of competent cells ===<br />
<html><br />
<object style="height: 170px; width: 270px" ><param name="movie" value="http://www.youtube.com/v/8fg_ldCMyW8" ><param name="allowFullScreen" value="true" ><param name="allowScriptAccess" value="always" ><embed src="http://www.youtube.com/v/8fg_ldCMyW8" type="application/x-shockwave-flash" allowfullscreen="true" allowScriptAccess="always" width="270" height="170" ></object><br />
</html><br><br />
Transformation is the process of introducing foreign DNA (e.g plasmids, BAC) into a bacterium. Bacterial cells intTransformation of competent cellso which foreign DNA can be transformed are called competent. Some bacteria are naturally competent (e.g B. subtilis), whereas others such as E. coli are not naturally competent. Non-competent cells can be made competent and then transformed via one of two main approaches; chemical transformation and electroporation. It is important to note we have tested transformations of the distribution kit with this protocol.[https://2010.igem.org/Team:Debrecen-Hungary/protocols/Transformation_of_competent_cells Read more...]<br />
<br />
[[Team:Debrecen-Hungary/protocols#Notebook|[TOP]]]<br />
<br />
=== Mini Prep ===<br />
This protocol is designed for purification of up to 20 μg of high-copy plasmid DNA from 1–5 ml overnight cultures of E. coli in LB (Luria-Bertani) medium [https://2010.igem.org/Team:Debrecen-Hungary/protocols/miniprep Read more...]<br />
<br />
===Midi Prep===<br />
<html><br />
<object style="height: 170px; width: 270px" ><param name="movie" value="http://www.youtube.com/v/4JyHe5gxihU" ><param name="allowFullScreen" value="true" ><param name="allowScriptAccess" value="always" ><embed src="http://www.youtube.com/v/4JyHe5gxihU" type="application/x-shockwave-flash" allowfullscreen="true" allowScriptAccess="always" width="270" height="170" ></object><br />
</html><br />
<br />
[https://2010.igem.org/Team:Debrecen-Hungary/protocols/midiprep Read more...]<br />
<br />
[[Team:Debrecen-Hungary/protocols#Notebook|[TOP]]]<br />
<br />
== Notebook protocols utilized by the molecular tools subteam ==<br />
<br />
=== Restriction enzyme digestion ===<br />
<br />
BioBrick standard biological parts are flanked by well characterized upstream and downstream sequences which are technically not considered part of the BioBrick part (aka prefix and suffix). These up/down stream segments contain restriction sites for specific restriction enzymes, which allows for the simple creation of larger BioBrick parts by chaining together smaller ones in any desired order.<br />
[https://2010.igem.org/Team:Debrecen-Hungary/protocols/Restriction_biobrick_parts Read more...]<br />
<br />
[[Team:Debrecen-Hungary/protocols#Notebook|[TOP]]]<br />
<br />
=== Excise agarose gel band for further purification and subcloning ===<br />
<html><br />
<object style="height: 170px; width: 270px" ><param name="movie" value="http://www.youtube.com/v/U74NaSx3rDY" ><param name="allowFullScreen" value="true" ><param name="allowScriptAccess" value="always" ><embed src="http://www.youtube.com/v/U74NaSx3rDY" type="application/x-shockwave-flash" allowfullscreen="true" allowScriptAccess="always" width="270" height="170" ></object><br />
</html><br />
<br />
[https://2010.igem.org/Team:Debrecen-Hungary/protocols/PCR_purification_from_Agarose_Gel Read more...]<br />
<br />
[[Team:Debrecen-Hungary/protocols#Notebook|[TOP]]]<br />
<br />
=== PCR ===<br />
<html><br />
<object style="height: 170px; width: 270px" ><param name="movie" value="http://www.youtube.com/v/vIbKpzlOAXA" ><param name="allowFullScreen" value="true" ><param name="allowScriptAccess" value="always" ><embed src="http://www.youtube.com/v/vIbKpzlOAXA" type="application/x-shockwave-flash" allowfullscreen="true" allowScriptAccess="always" width="270" height="170" ></object><br />
</html><br />
<br />
[https://2010.igem.org/Team:Debrecen-Hungary/protocols/PCR Read more...]<br />
<br />
[[Team:Debrecen-Hungary/protocols#Notebook|[TOP]]]<br />
<br />
=== Gel electrophoresis ===<br />
<html><br />
<object style="height: 170px; width: 270px" ><param name="movie" value="http://www.youtube.com/v/Q-kmMidAo3A" ><param name="allowFullScreen" value="true" ><param name="allowScriptAccess" value="always" ><embed src="http://www.youtube.com/v/Q-kmMidAo3A" type="application/x-shockwave-flash" allowfullscreen="true" allowScriptAccess="always" width="270" height="170" ></object><br />
</html><br />
<br />
[https://2010.igem.org/Team:Debrecen-Hungary/protocols/Gel_electrophoresis Read more...]<br />
<br />
[[Team:Debrecen-Hungary/protocols#Notebook|[TOP]]]<br />
<br />
== Notebook protocols utilized by the tissue culture subteam ==<br />
<br />
=== Cell Passaging ===<br />
<html><br />
<object style="height: 170px; width: 270px" ><param name="movie" value="http://www.youtube.com/v/7BniRI8C1PM" ><param name="allowFullScreen" value="true" ><param name="allowScriptAccess" value="always" ><embed src="http://www.youtube.com/v/7BniRI8C1PM" type="application/x-shockwave-flash" allowfullscreen="true" allowScriptAccess="always" width="270" height="170" ></object><br />
</html><br><br />
Cell passaging or splitting is a technique that enables an individual to keep cells alive and growing under cultured conditions for extended periods of time. Cells should be passed when they are 90%-100% confluent. You have to do the cell passage on every second to fourth day (i.e. on every Monday, Wednesday and Friday).<br />
After reaching the confluency, the cells do not get enough nutrients and do not have enough place where they can extend. The colour of the medium switches from reddish-pink to orange or yellow which shows acidic metabolic products.<br />
[https://2010.igem.org/Team:Debrecen-Hungary/protocols/Cell_Passaging Read more...]<br />
<br />
[[Team:Debrecen-Hungary/protocols#Notebook|[TOP]]]<br />
<br />
=== Preparation of DMEM Medium and PEI ===<br />
Eukaryotic cells, derived from multicellular animal eukaryotes, can be maintained in culturing medias. Aside from temperature and gas mixture, the most commonly varied factor in eucaryotic culture systems is the growth medium. Recipes for growth media can vary in pH, glucose concentration, growth factors, and the presence of other nutrients. The type of the media used depends on the type of the cell line. [https://2010.igem.org/Team:Debrecen-Hungary/protocols/_Media_PEI_Preparation Read more...]<br />
<br />
=== Transfection ===<br />
<html><br />
<object style="height: 170px; width: 270px" ><param name="movie" value="http://www.youtube.com/v/r8DlyOCRJRs" ><param name="allowFullScreen" value="true" ><param name="allowScriptAccess" value="always" ><embed src="http://www.youtube.com/v/r8DlyOCRJRs" type="application/x-shockwave-flash" allowfullscreen="true" allowScriptAccess="always" width="270" height="170" ></object><br />
</html><br />
<br />
The following protocol is used to succesfully introduce foreign plasmid DNA into pluripotent cells through a chemical way, fuGENE mediated transfection. [https://2010.igem.org/Team:Debrecen-Hungary/protocols/_Transfection Read more...]<br />
<br />
[[Team:Debrecen-Hungary/protocols#Notebook|[TOP]]]<br />
<br />
=== Ligand Treatment ===<br />
<html><br />
<object style="height: 170px; width: 270px" ><param name="movie" value="http://www.youtube.com/v/XnNfkADMjA0" ><param name="allowFullScreen" value="true" ><param name="allowScriptAccess" value="always" ><embed src="http://www.youtube.com/v/XnNfkADMjA0" type="application/x-shockwave-flash" allowfullscreen="true" allowScriptAccess="always" width="270" height="170" ></object><br />
</html><br><br />
Ligand treatment is a procedure when we add the appropriate ligand to the nuclear receptor (NR, transcription factor). The NR got into the cells through a previous transfection step. After treatment the ligand-binded NRs will dimerize and bind to the DNA at specific Nuclear receptor Response Elements and this will promote the gene expression of the downstream gene. In further examinations we detect the expression level of the target gene by Luciferase assay. [https://2010.igem.org/Team:Debrecen-Hungary/protocols/Ligand_Treatment Read more...]<br />
<br />
[[Team:Debrecen-Hungary/protocols#Notebook|[TOP]]]<br />
<br />
== Notebook protocols utilized by the Luciferase subteam ==<br />
<br />
=== Measuring Luciferase activity with the Victor plate reader ===<br />
<br />
he Wallac 1420 VICTOR2 is a multilabel, multitask plate reader designed to support the future demands of industrial and academic laboratories for multiple assay technologies on a single platform. An extended version of the successful Wallac VICTOR multilabel reader, the VICTOR2 allows immediate access to more than 10 counting modes, covering all of the main nonradioactive counting technologies. [https://2010.igem.org/Team:Debrecen-Hungary/protocols/_Enzymatic_activity_with_Victor_machine Read more...]<br />
<br />
[[Team:Debrecen-Hungary/protocols#Notebook|[TOP]]]<br />
<br />
==Online References At Openwetware==<br />
<br />
[https://2010.igem.org/Team:Debrecen-Hungary/protocols/makinglb ''Main article - Making Lurea's Broth'']<br />
<br />
[http://www.openwetware.org/wiki/IGEM:University_of_Debrecen:Transformation_of_competent_cells ''Main article - Transformation of competent cells'']<br />
<br />
[http://openwetware.org/wiki/Editing_IGEM:University_of_Debrecen:_Mini_Prep ''Main article - Mini prep'']<br />
<br />
[http://openwetware.org/wiki/IGEM:University_of_Debrecen:Restriction_biobrick_parts#Notes ''Main article - Restriction enzyme digestion'']<br />
<br />
[http://www.openwetware.org/wiki/IGEM:University_of_Debrecen:PCR_purification_from_Agarose_Gel ''Main article - PCR product purification / Gel purification''] <br><br />
<br />
[http://www.openwetware.org/wiki/IGEM:University_of_Debrecen:_Polymerase_Chain_Reaction_%28PCR%29 ''Main article - PCR''] <br><br />
<br />
[http://openwetware.org/wiki/IGEM:University_of_Debrecen:Gel_electrophoresis ''Main article - Gel electrophoresis'']<br />
<br />
[http://www.openwetware.org/wiki/IGEM:University_of_Debrecen:Cell_Passaging ''Main article - Cell passaging'']<br />
<br />
[http://openwetware.org/wiki/IGEM:University_of_Debrecen:_Media_PEI_Preparation ''Main article - Media PEI preparation'']<br />
<br />
[http://openwetware.org/wiki/IGEM:University_of_Debrecen:_transfection ''Main article - Transfection'']<br />
<br />
[http://openwetware.org/wiki/IGEM:University_of_Debrecen:Ligand_Treatment ''Main article - Ligand treatment''] <br />
<br />
[http://openwetware.org/wiki/IGEM:University_of_Debrecen:_Enzymatic_activity_with_Victor_machine ''Main article - Measuring Luciferase activity with the Victor plate reader'']<br />
</div><br />
<br />
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<a href="https://2010.igem.org/Team:Debrecen-Hungary"><img src="https://static.igem.org/mediawiki/2010/9/96/Backbuttonbck.jpg"></a><br />
</center><br />
</html></div>Liorhttp://2010.igem.org/Team:Debrecen-Hungary/protocolsTeam:Debrecen-Hungary/protocols2010-10-27T12:06:39Z<p>Lior: /* Ligand Treatment */</p>
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<br />
=Welcome To Our Protocols=<br />
<br />
<h1> "Science is nothing else but the art of proper reproducibility. " </h1> <br><br />
The iGEM experience is not merely a project or a conference, but it was the way that most of our students got acquainted with the world of biological research laboratory. Pipettes, solutions, gels, electrodes, dishes and other scary machinery quickly filled our lives. <br />
From day one we saw the vast importance of teaching our students to keep a proper laboratory journal. As time passed and our project grew., and with growth sprouted the idea of keeping an electronic laboratory journal with texts and video’s depicting the proper way of doing our niche of science. And so, we present to you our combined effort a text and video version<br />
<br />
=='''Contents'''==<br />
<br />
'''Notebook protocols utilized by the bacterial work subteam '''<br />
<br />
[[Team:Debrecen-Hungary/protocols#Making Lurea's Broth|Making Lurea's Broth]] - [[Team:Debrecen-Hungary/protocols#Transformation of competent cells|Transformation of competent cells]] - [[Team:Debrecen-Hungary/protocols#Mini Prep|Mini Prep]] - [[Team:Debrecen-Hungary/protocols#Midi Prep|Midi Prep]]<br />
<br />
'''Notebook protocols utilized by the molecular tools subteam '''<br />
<br />
[[Team:Debrecen-Hungary/protocols#Restriction enzyme digestion|Restriction enzyme digestion]] - [[Team:Debrecen-Hungary/protocols#Cutting gel for PCR product purification |PCR Purification]] - [[Team:Debrecen-Hungary/protocols#PCR|PCR]] - [[Team:Debrecen-Hungary/protocols#Gel electrophoresis|Gel electrophoresis]] <br />
<br />
'''Notebook protocols utilized by the tissue culture subteam'''<br />
<br />
[[Team:Debrecen-Hungary/protocols#Cell Passaging|Cell Passaging]] - [[Team:Debrecen-Hungary/protocols#Media PEI Preparation |Media PEI Preparation ]] - [[Team:Debrecen-Hungary/protocols#Transfection|Transfection ]] - [[Team:Debrecen-Hungary/protocols#Ligand Treatment |Ligand Treatment ]]<br />
<br />
'''Notebook protocols utilized by the Luciferase subteam'''<br />
<br />
[[Team:Debrecen-Hungary/protocols#Measuring Luciferase activity with the Victor plate reader |Measuring Luciferase activity with the Victor plate reader ]]<br />
<br />
== Notebook protocols utilized by the bacterial work subteam ==<br />
<br />
=== Making Lurea's Broth===<br />
<br />
[https://2010.igem.org/Team:Debrecen-Hungary/protocols/makinglb Read more...]<br />
<br />
[[Team:Debrecen-Hungary/protocols#Notebook|[TOP]]]<br />
<br />
=== Transformation of competent cells ===<br />
<html><br />
<object style="height: 170px; width: 270px" ><param name="movie" value="http://www.youtube.com/v/8fg_ldCMyW8" ><param name="allowFullScreen" value="true" ><param name="allowScriptAccess" value="always" ><embed src="http://www.youtube.com/v/8fg_ldCMyW8" type="application/x-shockwave-flash" allowfullscreen="true" allowScriptAccess="always" width="270" height="170" ></object><br />
</html><br><br />
Transformation is the process of introducing foreign DNA (e.g plasmids, BAC) into a bacterium. Bacterial cells intTransformation of competent cellso which foreign DNA can be transformed are called competent. Some bacteria are naturally competent (e.g B. subtilis), whereas others such as E. coli are not naturally competent. Non-competent cells can be made competent and then transformed via one of two main approaches; chemical transformation and electroporation. It is important to note we have tested transformations of the distribution kit with this protocol.[https://2010.igem.org/Team:Debrecen-Hungary/protocols/Transformation_of_competent_cells Read more...]<br />
<br />
[[Team:Debrecen-Hungary/protocols#Notebook|[TOP]]]<br />
<br />
=== Mini Prep ===<br />
This protocol is designed for purification of up to 20 μg of high-copy plasmid DNA from 1–5 ml overnight cultures of E. coli in LB (Luria-Bertani) medium [https://2010.igem.org/Team:Debrecen-Hungary/protocols/miniprep Read more...]<br />
<br />
===Midi Prep===<br />
<html><br />
<object style="height: 170px; width: 270px" ><param name="movie" value="http://www.youtube.com/v/4JyHe5gxihU" ><param name="allowFullScreen" value="true" ><param name="allowScriptAccess" value="always" ><embed src="http://www.youtube.com/v/4JyHe5gxihU" type="application/x-shockwave-flash" allowfullscreen="true" allowScriptAccess="always" width="270" height="170" ></object><br />
</html><br />
<br />
[https://2010.igem.org/Team:Debrecen-Hungary/protocols/midiprep Read more...]<br />
<br />
[[Team:Debrecen-Hungary/protocols#Notebook|[TOP]]]<br />
<br />
== Notebook protocols utilized by the molecular tools subteam ==<br />
<br />
=== Restriction enzyme digestion ===<br />
<br />
BioBrick standard biological parts are flanked by well characterized upstream and downstream sequences which are technically not considered part of the BioBrick part (aka prefix and suffix). These up/down stream segments contain restriction sites for specific restriction enzymes, which allows for the simple creation of larger BioBrick parts by chaining together smaller ones in any desired order.<br />
[https://2010.igem.org/Team:Debrecen-Hungary/protocols/Restriction_biobrick_parts Read more...]<br />
<br />
[[Team:Debrecen-Hungary/protocols#Notebook|[TOP]]]<br />
<br />
=== Excise agarose gel band for further purification and subcloning ===<br />
<html><br />
<object style="height: 170px; width: 270px" ><param name="movie" value="http://www.youtube.com/v/U74NaSx3rDY" ><param name="allowFullScreen" value="true" ><param name="allowScriptAccess" value="always" ><embed src="http://www.youtube.com/v/U74NaSx3rDY" type="application/x-shockwave-flash" allowfullscreen="true" allowScriptAccess="always" width="270" height="170" ></object><br />
</html><br />
<br />
[https://2010.igem.org/Team:Debrecen-Hungary/protocols/PCR_purification_from_Agarose_Gel Read more...]<br />
<br />
[[Team:Debrecen-Hungary/protocols#Notebook|[TOP]]]<br />
<br />
=== PCR ===<br />
<html><br />
<object style="height: 170px; width: 270px" ><param name="movie" value="http://www.youtube.com/v/vIbKpzlOAXA" ><param name="allowFullScreen" value="true" ><param name="allowScriptAccess" value="always" ><embed src="http://www.youtube.com/v/vIbKpzlOAXA" type="application/x-shockwave-flash" allowfullscreen="true" allowScriptAccess="always" width="270" height="170" ></object><br />
</html><br />
<br />
[https://2010.igem.org/Team:Debrecen-Hungary/protocols/PCR Read more...]<br />
<br />
[[Team:Debrecen-Hungary/protocols#Notebook|[TOP]]]<br />
<br />
=== Gel electrophoresis ===<br />
<html><br />
<object style="height: 170px; width: 270px" ><param name="movie" value="http://www.youtube.com/v/Q-kmMidAo3A" ><param name="allowFullScreen" value="true" ><param name="allowScriptAccess" value="always" ><embed src="http://www.youtube.com/v/Q-kmMidAo3A" type="application/x-shockwave-flash" allowfullscreen="true" allowScriptAccess="always" width="270" height="170" ></object><br />
</html><br />
<br />
[https://2010.igem.org/Team:Debrecen-Hungary/protocols/Gel_electrophoresis Read more...]<br />
<br />
[[Team:Debrecen-Hungary/protocols#Notebook|[TOP]]]<br />
<br />
== Notebook protocols utilized by the tissue culture subteam ==<br />
<br />
=== Cell Passaging ===<br />
<html><br />
<object style="height: 170px; width: 270px" ><param name="movie" value="http://www.youtube.com/v/7BniRI8C1PM" ><param name="allowFullScreen" value="true" ><param name="allowScriptAccess" value="always" ><embed src="http://www.youtube.com/v/7BniRI8C1PM" type="application/x-shockwave-flash" allowfullscreen="true" allowScriptAccess="always" width="270" height="170" ></object><br />
</html><br><br />
Cell passaging or splitting is a technique that enables an individual to keep cells alive and growing under cultured conditions for extended periods of time. Cells should be passed when they are 90%-100% confluent. You have to do the cell passage on every second to fourth day (i.e. on every Monday, Wednesday and Friday).<br />
After reaching the confluency, the cells do not get enough nutrients and do not have enough place where they can extend. The colour of the medium switches from reddish-pink to orange or yellow which shows acidic metabolic products.<br />
[https://2010.igem.org/Team:Debrecen-Hungary/protocols/Cell_Passaging Read more...]<br />
<br />
[[Team:Debrecen-Hungary/protocols#Notebook|[TOP]]]<br />
<br />
=== Preparation of DMEM Medium and PEI ===<br />
Eukaryotic cells, derived from multicellular animal eukaryotes, can be maintained in culturing medias. Aside from temperature and gas mixture, the most commonly varied factor in eucaryotic culture systems is the growth medium. Recipes for growth media can vary in pH, glucose concentration, growth factors, and the presence of other nutrients. The type of the media used depends on the type of the cell line. [https://2010.igem.org/Team:Debrecen-Hungary/protocols/_Media_PEI_Preparation Read more...]<br />
<br />
=== Transfection ===<br />
<html><br />
<object style="height: 170px; width: 270px" ><param name="movie" value="http://www.youtube.com/v/r8DlyOCRJRs" ><param name="allowFullScreen" value="true" ><param name="allowScriptAccess" value="always" ><embed src="http://www.youtube.com/v/r8DlyOCRJRs" type="application/x-shockwave-flash" allowfullscreen="true" allowScriptAccess="always" width="270" height="170" ></object><br />
</html><br />
<br />
The following protocol is used to succesfully introduce foreign plasmid DNA into pluripotent cells through a chemical way, fuGENE mediated transfection. [https://2010.igem.org/Team:Debrecen-Hungary/protocols/_Transfection Read more...]<br />
<br />
[[Team:Debrecen-Hungary/protocols#Notebook|[TOP]]]<br />
<br />
=== Ligand Treatment ===<br />
<html><br />
<object style="height: 170px; width: 270px" ><param name="movie" value="http://www.youtube.com/v/XnNfkADMjA0" ><param name="allowFullScreen" value="true" ><param name="allowScriptAccess" value="always" ><embed src="http://www.youtube.com/v/XnNfkADMjA0" type="application/x-shockwave-flash" allowfullscreen="true" allowScriptAccess="always" width="270" height="170" ></object><br />
</html><br><br />
Ligand treatment is a procedure when we add the appropriate ligand to the nuclear receptor (NR, transcription factor). The NR got into the cells through a previous transfection step. After treatment the ligand-binded NRs will dimerize and bind to the DNA at specific Nuclear receptor Response Elements and this will promote the gene expression of the downstream gene. In further examinations we detect the expression level of the target gene by Luciferase assay. [https://2010.igem.org/Team:Debrecen-Hungary/protocols/Ligand_Treatment Read more...]<br />
<br />
[[Team:Debrecen-Hungary/protocols#Notebook|[TOP]]]<br />
<br />
== Notebook protocols utilized by the Luciferase subteam ==<br />
<br />
=== Measuring Luciferase activity with the Victor plate reader ===<br />
<br />
he Wallac 1420 VICTOR2 is a multilabel, multitask plate reader designed to support the future demands of industrial and academic laboratories for multiple assay technologies on a single platform. An extended version of the successful Wallac VICTOR multilabel reader, the VICTOR2 allows immediate access to more than 10 counting modes, covering all of the main nonradioactive counting technologies. [https://2010.igem.org/Team:Debrecen-Hungary/protocols/_Enzymatic_activity_with_Victor_machine Read more...]<br />
<br />
[[Team:Debrecen-Hungary/protocols#Notebook|[TOP]]]<br />
<br />
==Online References At Openwetware==<br />
<br />
[https://2010.igem.org/Team:Debrecen-Hungary/protocols/makinglb ''Main article - Making Lurea's Broth'']<br />
<br />
[http://www.openwetware.org/wiki/IGEM:University_of_Debrecen:Transformation_of_competent_cells ''Main article - Transformation of competent cells'']<br />
<br />
[http://openwetware.org/wiki/Editing_IGEM:University_of_Debrecen:_Mini_Prep ''Main article - Mini prep'']<br />
<br />
[http://openwetware.org/wiki/IGEM:University_of_Debrecen:Restriction_biobrick_parts#Notes ''Main article - Restriction enzyme digestion'']<br />
<br />
[http://www.openwetware.org/wiki/IGEM:University_of_Debrecen:PCR_purification_from_Agarose_Gel ''Main article - PCR product purification / Gel purification''] <br><br />
<br />
[http://www.openwetware.org/wiki/IGEM:University_of_Debrecen:_Polymerase_Chain_Reaction_%28PCR%29 ''Main article - PCR''] <br><br />
<br />
[http://openwetware.org/wiki/IGEM:University_of_Debrecen:Gel_electrophoresis ''Main article - Gel electrophoresis'']<br />
<br />
[http://www.openwetware.org/wiki/IGEM:University_of_Debrecen:Cell_Passaging ''Main article - Cell passaging'']<br />
<br />
[http://openwetware.org/wiki/IGEM:University_of_Debrecen:_Media_PEI_Preparation ''Main article - Media PEI preparation'']<br />
<br />
[http://www.openwetware.org/wiki/IGEM:University_of_Debrecen:_Transfection ''Main article - Transfection'']<br />
<br />
[http://openwetware.org/wiki/IGEM:University_of_Debrecen:Ligand_Treatment ''Main article - Ligand treatment''] <br />
<br />
[http://openwetware.org/wiki/IGEM:University_of_Debrecen:_Enzymatic_activity_with_Victor_machine ''Main article - Measuring Luciferase activity with the Victor plate reader'']<br />
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<a href="https://2010.igem.org/Team:Debrecen-Hungary"><img src="https://static.igem.org/mediawiki/2010/9/96/Backbuttonbck.jpg"></a><br />
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</html></div>Liorhttp://2010.igem.org/Team:Debrecen-Hungary/protocolsTeam:Debrecen-Hungary/protocols2010-10-27T12:06:19Z<p>Lior: /* Transfection */</p>
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<br />
=Welcome To Our Protocols=<br />
<br />
<h1> "Science is nothing else but the art of proper reproducibility. " </h1> <br><br />
The iGEM experience is not merely a project or a conference, but it was the way that most of our students got acquainted with the world of biological research laboratory. Pipettes, solutions, gels, electrodes, dishes and other scary machinery quickly filled our lives. <br />
From day one we saw the vast importance of teaching our students to keep a proper laboratory journal. As time passed and our project grew., and with growth sprouted the idea of keeping an electronic laboratory journal with texts and video’s depicting the proper way of doing our niche of science. And so, we present to you our combined effort a text and video version<br />
<br />
=='''Contents'''==<br />
<br />
'''Notebook protocols utilized by the bacterial work subteam '''<br />
<br />
[[Team:Debrecen-Hungary/protocols#Making Lurea's Broth|Making Lurea's Broth]] - [[Team:Debrecen-Hungary/protocols#Transformation of competent cells|Transformation of competent cells]] - [[Team:Debrecen-Hungary/protocols#Mini Prep|Mini Prep]] - [[Team:Debrecen-Hungary/protocols#Midi Prep|Midi Prep]]<br />
<br />
'''Notebook protocols utilized by the molecular tools subteam '''<br />
<br />
[[Team:Debrecen-Hungary/protocols#Restriction enzyme digestion|Restriction enzyme digestion]] - [[Team:Debrecen-Hungary/protocols#Cutting gel for PCR product purification |PCR Purification]] - [[Team:Debrecen-Hungary/protocols#PCR|PCR]] - [[Team:Debrecen-Hungary/protocols#Gel electrophoresis|Gel electrophoresis]] <br />
<br />
'''Notebook protocols utilized by the tissue culture subteam'''<br />
<br />
[[Team:Debrecen-Hungary/protocols#Cell Passaging|Cell Passaging]] - [[Team:Debrecen-Hungary/protocols#Media PEI Preparation |Media PEI Preparation ]] - [[Team:Debrecen-Hungary/protocols#Transfection|Transfection ]] - [[Team:Debrecen-Hungary/protocols#Ligand Treatment |Ligand Treatment ]]<br />
<br />
'''Notebook protocols utilized by the Luciferase subteam'''<br />
<br />
[[Team:Debrecen-Hungary/protocols#Measuring Luciferase activity with the Victor plate reader |Measuring Luciferase activity with the Victor plate reader ]]<br />
<br />
== Notebook protocols utilized by the bacterial work subteam ==<br />
<br />
=== Making Lurea's Broth===<br />
<br />
[https://2010.igem.org/Team:Debrecen-Hungary/protocols/makinglb Read more...]<br />
<br />
[[Team:Debrecen-Hungary/protocols#Notebook|[TOP]]]<br />
<br />
=== Transformation of competent cells ===<br />
<html><br />
<object style="height: 170px; width: 270px" ><param name="movie" value="http://www.youtube.com/v/8fg_ldCMyW8" ><param name="allowFullScreen" value="true" ><param name="allowScriptAccess" value="always" ><embed src="http://www.youtube.com/v/8fg_ldCMyW8" type="application/x-shockwave-flash" allowfullscreen="true" allowScriptAccess="always" width="270" height="170" ></object><br />
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Transformation is the process of introducing foreign DNA (e.g plasmids, BAC) into a bacterium. Bacterial cells intTransformation of competent cellso which foreign DNA can be transformed are called competent. Some bacteria are naturally competent (e.g B. subtilis), whereas others such as E. coli are not naturally competent. Non-competent cells can be made competent and then transformed via one of two main approaches; chemical transformation and electroporation. It is important to note we have tested transformations of the distribution kit with this protocol.[https://2010.igem.org/Team:Debrecen-Hungary/protocols/Transformation_of_competent_cells Read more...]<br />
<br />
[[Team:Debrecen-Hungary/protocols#Notebook|[TOP]]]<br />
<br />
=== Mini Prep ===<br />
This protocol is designed for purification of up to 20 μg of high-copy plasmid DNA from 1–5 ml overnight cultures of E. coli in LB (Luria-Bertani) medium [https://2010.igem.org/Team:Debrecen-Hungary/protocols/miniprep Read more...]<br />
<br />
===Midi Prep===<br />
<html><br />
<object style="height: 170px; width: 270px" ><param name="movie" value="http://www.youtube.com/v/4JyHe5gxihU" ><param name="allowFullScreen" value="true" ><param name="allowScriptAccess" value="always" ><embed src="http://www.youtube.com/v/4JyHe5gxihU" type="application/x-shockwave-flash" allowfullscreen="true" allowScriptAccess="always" width="270" height="170" ></object><br />
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<br />
[https://2010.igem.org/Team:Debrecen-Hungary/protocols/midiprep Read more...]<br />
<br />
[[Team:Debrecen-Hungary/protocols#Notebook|[TOP]]]<br />
<br />
== Notebook protocols utilized by the molecular tools subteam ==<br />
<br />
=== Restriction enzyme digestion ===<br />
<br />
BioBrick standard biological parts are flanked by well characterized upstream and downstream sequences which are technically not considered part of the BioBrick part (aka prefix and suffix). These up/down stream segments contain restriction sites for specific restriction enzymes, which allows for the simple creation of larger BioBrick parts by chaining together smaller ones in any desired order.<br />
[https://2010.igem.org/Team:Debrecen-Hungary/protocols/Restriction_biobrick_parts Read more...]<br />
<br />
[[Team:Debrecen-Hungary/protocols#Notebook|[TOP]]]<br />
<br />
=== Excise agarose gel band for further purification and subcloning ===<br />
<html><br />
<object style="height: 170px; width: 270px" ><param name="movie" value="http://www.youtube.com/v/U74NaSx3rDY" ><param name="allowFullScreen" value="true" ><param name="allowScriptAccess" value="always" ><embed src="http://www.youtube.com/v/U74NaSx3rDY" type="application/x-shockwave-flash" allowfullscreen="true" allowScriptAccess="always" width="270" height="170" ></object><br />
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<br />
[https://2010.igem.org/Team:Debrecen-Hungary/protocols/PCR_purification_from_Agarose_Gel Read more...]<br />
<br />
[[Team:Debrecen-Hungary/protocols#Notebook|[TOP]]]<br />
<br />
=== PCR ===<br />
<html><br />
<object style="height: 170px; width: 270px" ><param name="movie" value="http://www.youtube.com/v/vIbKpzlOAXA" ><param name="allowFullScreen" value="true" ><param name="allowScriptAccess" value="always" ><embed src="http://www.youtube.com/v/vIbKpzlOAXA" type="application/x-shockwave-flash" allowfullscreen="true" allowScriptAccess="always" width="270" height="170" ></object><br />
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[https://2010.igem.org/Team:Debrecen-Hungary/protocols/PCR Read more...]<br />
<br />
[[Team:Debrecen-Hungary/protocols#Notebook|[TOP]]]<br />
<br />
=== Gel electrophoresis ===<br />
<html><br />
<object style="height: 170px; width: 270px" ><param name="movie" value="http://www.youtube.com/v/Q-kmMidAo3A" ><param name="allowFullScreen" value="true" ><param name="allowScriptAccess" value="always" ><embed src="http://www.youtube.com/v/Q-kmMidAo3A" type="application/x-shockwave-flash" allowfullscreen="true" allowScriptAccess="always" width="270" height="170" ></object><br />
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[https://2010.igem.org/Team:Debrecen-Hungary/protocols/Gel_electrophoresis Read more...]<br />
<br />
[[Team:Debrecen-Hungary/protocols#Notebook|[TOP]]]<br />
<br />
== Notebook protocols utilized by the tissue culture subteam ==<br />
<br />
=== Cell Passaging ===<br />
<html><br />
<object style="height: 170px; width: 270px" ><param name="movie" value="http://www.youtube.com/v/7BniRI8C1PM" ><param name="allowFullScreen" value="true" ><param name="allowScriptAccess" value="always" ><embed src="http://www.youtube.com/v/7BniRI8C1PM" type="application/x-shockwave-flash" allowfullscreen="true" allowScriptAccess="always" width="270" height="170" ></object><br />
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Cell passaging or splitting is a technique that enables an individual to keep cells alive and growing under cultured conditions for extended periods of time. Cells should be passed when they are 90%-100% confluent. You have to do the cell passage on every second to fourth day (i.e. on every Monday, Wednesday and Friday).<br />
After reaching the confluency, the cells do not get enough nutrients and do not have enough place where they can extend. The colour of the medium switches from reddish-pink to orange or yellow which shows acidic metabolic products.<br />
[https://2010.igem.org/Team:Debrecen-Hungary/protocols/Cell_Passaging Read more...]<br />
<br />
[[Team:Debrecen-Hungary/protocols#Notebook|[TOP]]]<br />
<br />
=== Preparation of DMEM Medium and PEI ===<br />
Eukaryotic cells, derived from multicellular animal eukaryotes, can be maintained in culturing medias. Aside from temperature and gas mixture, the most commonly varied factor in eucaryotic culture systems is the growth medium. Recipes for growth media can vary in pH, glucose concentration, growth factors, and the presence of other nutrients. The type of the media used depends on the type of the cell line. [https://2010.igem.org/Team:Debrecen-Hungary/protocols/_Media_PEI_Preparation Read more...]<br />
<br />
=== Transfection ===<br />
<html><br />
<object style="height: 170px; width: 270px" ><param name="movie" value="http://www.youtube.com/v/r8DlyOCRJRs" ><param name="allowFullScreen" value="true" ><param name="allowScriptAccess" value="always" ><embed src="http://www.youtube.com/v/r8DlyOCRJRs" type="application/x-shockwave-flash" allowfullscreen="true" allowScriptAccess="always" width="270" height="170" ></object><br />
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<br />
The following protocol is used to succesfully introduce foreign plasmid DNA into pluripotent cells through a chemical way, fuGENE mediated transfection. [https://2010.igem.org/Team:Debrecen-Hungary/protocols/_Transfection Read more...]<br />
<br />
[[Team:Debrecen-Hungary/protocols#Notebook|[TOP]]]<br />
<br />
=== Ligand Treatment ===<br />
<html><br />
<object style="height: 170px; width: 270px" ><param name="movie" value="http://www.youtube.com/v/XnNfkADMjA0" ><param name="allowFullScreen" value="true" ><param name="allowScriptAccess" value="always" ><embed src="http://www.youtube.com/v/XnNfkADMjA0" type="application/x-shockwave-flash" allowfullscreen="true" allowScriptAccess="always" width="270" height="170" ></object><br />
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Ligand treatment is a procedure when we add the appropriate ligand to the nuclear receptor (NR, transcription factor). The NR got into the cells through a previous transfection step. After treatment the ligand-binded NRs will dimerize and bind to the DNA at specific Nuclear receptor Response Elements and this will promote the gene expression of the downstream gene. In further examinations we detect the expression level of the target gene by Luciferase assay. [https://2010.igem.org/Team:Debrecen-Hungary/protocols/Ligand_Treatment Read more...]<br />
<br />
[[Team:Debrecen-Hungary/protocols#Notebook|[TOP]]]<br />
<br />
== Notebook protocols utilized by the Luciferase subteam ==<br />
<br />
=== Measuring Luciferase activity with the Victor plate reader ===<br />
<br />
he Wallac 1420 VICTOR2 is a multilabel, multitask plate reader designed to support the future demands of industrial and academic laboratories for multiple assay technologies on a single platform. An extended version of the successful Wallac VICTOR multilabel reader, the VICTOR2 allows immediate access to more than 10 counting modes, covering all of the main nonradioactive counting technologies. [https://2010.igem.org/Team:Debrecen-Hungary/protocols/_Enzymatic_activity_with_Victor_machine Read more...]<br />
<br />
[[Team:Debrecen-Hungary/protocols#Notebook|[TOP]]]<br />
<br />
==Online References At Openwetware==<br />
<br />
[https://2010.igem.org/Team:Debrecen-Hungary/protocols/makinglb ''Main article - Making Lurea's Broth'']<br />
<br />
[http://www.openwetware.org/wiki/IGEM:University_of_Debrecen:Transformation_of_competent_cells ''Main article - Transformation of competent cells'']<br />
<br />
[http://openwetware.org/wiki/Editing_IGEM:University_of_Debrecen:_Mini_Prep ''Main article - Mini prep'']<br />
<br />
[http://openwetware.org/wiki/IGEM:University_of_Debrecen:Restriction_biobrick_parts#Notes ''Main article - Restriction enzyme digestion'']<br />
<br />
[http://www.openwetware.org/wiki/IGEM:University_of_Debrecen:PCR_purification_from_Agarose_Gel ''Main article - PCR product purification / Gel purification''] <br><br />
<br />
[http://www.openwetware.org/wiki/IGEM:University_of_Debrecen:_Polymerase_Chain_Reaction_%28PCR%29 ''Main article - PCR''] <br><br />
<br />
[http://openwetware.org/wiki/IGEM:University_of_Debrecen:Gel_electrophoresis ''Main article - Gel electrophoresis'']<br />
<br />
[http://www.openwetware.org/wiki/IGEM:University_of_Debrecen:Cell_Passaging ''Main article - Cell passaging'']<br />
<br />
[http://openwetware.org/wiki/IGEM:University_of_Debrecen:_Media_PEI_Preparation ''Main article - Media PEI preparation'']<br />
<br />
[http://www.openwetware.org/wiki/IGEM:University_of_Debrecen:_Transfection ''Main article - Transfection'']<br />
<br />
[http://openwetware.org/wiki/IGEM:University_of_Debrecen:Ligand_Treatment ''Main article - Ligand treatment''] <br />
<br />
[http://openwetware.org/wiki/IGEM:University_of_Debrecen:_Enzymatic_activity_with_Victor_machine ''Main article - Measuring Luciferase activity with the Victor plate reader'']<br />
</div><br />
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<a href="https://2010.igem.org/Team:Debrecen-Hungary"><img src="https://static.igem.org/mediawiki/2010/9/96/Backbuttonbck.jpg"></a><br />
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</html></div>Liorhttp://2010.igem.org/Team:Debrecen-Hungary/protocolsTeam:Debrecen-Hungary/protocols2010-10-27T12:02:21Z<p>Lior: /* Notebook protocols utilized by the molecular tools subteam */</p>
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<img src="https://static.igem.org/mediawiki/2010/3/3a/Protocols.jpg" width="400" height="110"><br />
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<a href="https://2010.igem.org/Team:Debrecen-Hungary"><img src="https://static.igem.org/mediawiki/2010/9/96/Backbuttonbck.jpg" align="right"></a><br />
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<body id="Notebook"></body></html><br />
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<div align="justify"><br />
<br />
<br />
<br />
=Welcome To Our Protocols=<br />
<br />
<h1> "Science is nothing else but the art of proper reproducibility. " </h1> <br><br />
The iGEM experience is not merely a project or a conference, but it was the way that most of our students got acquainted with the world of biological research laboratory. Pipettes, solutions, gels, electrodes, dishes and other scary machinery quickly filled our lives. <br />
From day one we saw the vast importance of teaching our students to keep a proper laboratory journal. As time passed and our project grew., and with growth sprouted the idea of keeping an electronic laboratory journal with texts and video’s depicting the proper way of doing our niche of science. And so, we present to you our combined effort a text and video version<br />
<br />
=='''Contents'''==<br />
<br />
'''Notebook protocols utilized by the bacterial work subteam '''<br />
<br />
[[Team:Debrecen-Hungary/protocols#Making Lurea's Broth|Making Lurea's Broth]] - [[Team:Debrecen-Hungary/protocols#Transformation of competent cells|Transformation of competent cells]] - [[Team:Debrecen-Hungary/protocols#Mini Prep|Mini Prep]] - [[Team:Debrecen-Hungary/protocols#Midi Prep|Midi Prep]]<br />
<br />
'''Notebook protocols utilized by the molecular tools subteam '''<br />
<br />
[[Team:Debrecen-Hungary/protocols#Restriction enzyme digestion|Restriction enzyme digestion]] - [[Team:Debrecen-Hungary/protocols#Cutting gel for PCR product purification |PCR Purification]] - [[Team:Debrecen-Hungary/protocols#PCR|PCR]] - [[Team:Debrecen-Hungary/protocols#Gel electrophoresis|Gel electrophoresis]] <br />
<br />
'''Notebook protocols utilized by the tissue culture subteam'''<br />
<br />
[[Team:Debrecen-Hungary/protocols#Cell Passaging|Cell Passaging]] - [[Team:Debrecen-Hungary/protocols#Media PEI Preparation |Media PEI Preparation ]] - [[Team:Debrecen-Hungary/protocols#Transfection|Transfection ]] - [[Team:Debrecen-Hungary/protocols#Ligand Treatment |Ligand Treatment ]]<br />
<br />
'''Notebook protocols utilized by the Luciferase subteam'''<br />
<br />
[[Team:Debrecen-Hungary/protocols#Measuring Luciferase activity with the Victor plate reader |Measuring Luciferase activity with the Victor plate reader ]]<br />
<br />
== Notebook protocols utilized by the bacterial work subteam ==<br />
<br />
=== Making Lurea's Broth===<br />
<br />
[https://2010.igem.org/Team:Debrecen-Hungary/protocols/makinglb Read more...]<br />
<br />
[[Team:Debrecen-Hungary/protocols#Notebook|[TOP]]]<br />
<br />
=== Transformation of competent cells ===<br />
<html><br />
<object style="height: 170px; width: 270px" ><param name="movie" value="http://www.youtube.com/v/8fg_ldCMyW8" ><param name="allowFullScreen" value="true" ><param name="allowScriptAccess" value="always" ><embed src="http://www.youtube.com/v/8fg_ldCMyW8" type="application/x-shockwave-flash" allowfullscreen="true" allowScriptAccess="always" width="270" height="170" ></object><br />
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Transformation is the process of introducing foreign DNA (e.g plasmids, BAC) into a bacterium. Bacterial cells intTransformation of competent cellso which foreign DNA can be transformed are called competent. Some bacteria are naturally competent (e.g B. subtilis), whereas others such as E. coli are not naturally competent. Non-competent cells can be made competent and then transformed via one of two main approaches; chemical transformation and electroporation. It is important to note we have tested transformations of the distribution kit with this protocol.[https://2010.igem.org/Team:Debrecen-Hungary/protocols/Transformation_of_competent_cells Read more...]<br />
<br />
[[Team:Debrecen-Hungary/protocols#Notebook|[TOP]]]<br />
<br />
=== Mini Prep ===<br />
This protocol is designed for purification of up to 20 μg of high-copy plasmid DNA from 1–5 ml overnight cultures of E. coli in LB (Luria-Bertani) medium [https://2010.igem.org/Team:Debrecen-Hungary/protocols/miniprep Read more...]<br />
<br />
===Midi Prep===<br />
<html><br />
<object style="height: 170px; width: 270px" ><param name="movie" value="http://www.youtube.com/v/4JyHe5gxihU" ><param name="allowFullScreen" value="true" ><param name="allowScriptAccess" value="always" ><embed src="http://www.youtube.com/v/4JyHe5gxihU" type="application/x-shockwave-flash" allowfullscreen="true" allowScriptAccess="always" width="270" height="170" ></object><br />
</html><br />
<br />
[https://2010.igem.org/Team:Debrecen-Hungary/protocols/midiprep Read more...]<br />
<br />
[[Team:Debrecen-Hungary/protocols#Notebook|[TOP]]]<br />
<br />
== Notebook protocols utilized by the molecular tools subteam ==<br />
<br />
=== Restriction enzyme digestion ===<br />
<br />
BioBrick standard biological parts are flanked by well characterized upstream and downstream sequences which are technically not considered part of the BioBrick part (aka prefix and suffix). These up/down stream segments contain restriction sites for specific restriction enzymes, which allows for the simple creation of larger BioBrick parts by chaining together smaller ones in any desired order.<br />
[https://2010.igem.org/Team:Debrecen-Hungary/protocols/Restriction_biobrick_parts Read more...]<br />
<br />
[[Team:Debrecen-Hungary/protocols#Notebook|[TOP]]]<br />
<br />
=== Excise agarose gel band for further purification and subcloning ===<br />
<html><br />
<object style="height: 170px; width: 270px" ><param name="movie" value="http://www.youtube.com/v/U74NaSx3rDY" ><param name="allowFullScreen" value="true" ><param name="allowScriptAccess" value="always" ><embed src="http://www.youtube.com/v/U74NaSx3rDY" type="application/x-shockwave-flash" allowfullscreen="true" allowScriptAccess="always" width="270" height="170" ></object><br />
</html><br />
<br />
[https://2010.igem.org/Team:Debrecen-Hungary/protocols/PCR_purification_from_Agarose_Gel Read more...]<br />
<br />
[[Team:Debrecen-Hungary/protocols#Notebook|[TOP]]]<br />
<br />
=== PCR ===<br />
<html><br />
<object style="height: 170px; width: 270px" ><param name="movie" value="http://www.youtube.com/v/vIbKpzlOAXA" ><param name="allowFullScreen" value="true" ><param name="allowScriptAccess" value="always" ><embed src="http://www.youtube.com/v/vIbKpzlOAXA" type="application/x-shockwave-flash" allowfullscreen="true" allowScriptAccess="always" width="270" height="170" ></object><br />
</html><br />
<br />
[https://2010.igem.org/Team:Debrecen-Hungary/protocols/PCR Read more...]<br />
<br />
[[Team:Debrecen-Hungary/protocols#Notebook|[TOP]]]<br />
<br />
=== Gel electrophoresis ===<br />
<html><br />
<object style="height: 170px; width: 270px" ><param name="movie" value="http://www.youtube.com/v/Q-kmMidAo3A" ><param name="allowFullScreen" value="true" ><param name="allowScriptAccess" value="always" ><embed src="http://www.youtube.com/v/Q-kmMidAo3A" type="application/x-shockwave-flash" allowfullscreen="true" allowScriptAccess="always" width="270" height="170" ></object><br />
</html><br />
<br />
[https://2010.igem.org/Team:Debrecen-Hungary/protocols/Gel_electrophoresis Read more...]<br />
<br />
[[Team:Debrecen-Hungary/protocols#Notebook|[TOP]]]<br />
<br />
== Notebook protocols utilized by the tissue culture subteam ==<br />
<br />
=== Cell Passaging ===<br />
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Cell passaging or splitting is a technique that enables an individual to keep cells alive and growing under cultured conditions for extended periods of time. Cells should be passed when they are 90%-100% confluent. You have to do the cell passage on every second to fourth day (i.e. on every Monday, Wednesday and Friday).<br />
After reaching the confluency, the cells do not get enough nutrients and do not have enough place where they can extend. The colour of the medium switches from reddish-pink to orange or yellow which shows acidic metabolic products.<br />
[https://2010.igem.org/Team:Debrecen-Hungary/protocols/Cell_Passaging Read more...]<br />
<br />
[[Team:Debrecen-Hungary/protocols#Notebook|[TOP]]]<br />
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=== Preparation of DMEM Medium and PEI ===<br />
Eukaryotic cells, derived from multicellular animal eukaryotes, can be maintained in culturing medias. Aside from temperature and gas mixture, the most commonly varied factor in eucaryotic culture systems is the growth medium. Recipes for growth media can vary in pH, glucose concentration, growth factors, and the presence of other nutrients. The type of the media used depends on the type of the cell line. [https://2010.igem.org/Team:Debrecen-Hungary/protocols/_Media_PEI_Preparation Read more...]<br />
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=== Transfection ===<br />
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The following protocol is used to succesfully introduce foreign plasmid DNA into pluripotent cells through a chemical way, PEI mediated transfection. PEI stands for Poliethilenimine, a cationic polymer which can bind nucleic acids and helps the DNA to get into the cells by receptor-mediated endocytosis.<br />
[https://2010.igem.org/Team:Debrecen-Hungary/protocols/_Transfection Read more...]<br />
<br />
[[Team:Debrecen-Hungary/protocols#Notebook|[TOP]]]<br />
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=== Ligand Treatment ===<br />
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Ligand treatment is a procedure when we add the appropriate ligand to the nuclear receptor (NR, transcription factor). The NR got into the cells through a previous transfection step. After treatment the ligand-binded NRs will dimerize and bind to the DNA at specific Nuclear receptor Response Elements and this will promote the gene expression of the downstream gene. In further examinations we detect the expression level of the target gene by Luciferase assay. [https://2010.igem.org/Team:Debrecen-Hungary/protocols/Ligand_Treatment Read more...]<br />
<br />
[[Team:Debrecen-Hungary/protocols#Notebook|[TOP]]]<br />
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== Notebook protocols utilized by the Luciferase subteam ==<br />
<br />
=== Measuring Luciferase activity with the Victor plate reader ===<br />
<br />
he Wallac 1420 VICTOR2 is a multilabel, multitask plate reader designed to support the future demands of industrial and academic laboratories for multiple assay technologies on a single platform. An extended version of the successful Wallac VICTOR multilabel reader, the VICTOR2 allows immediate access to more than 10 counting modes, covering all of the main nonradioactive counting technologies. [https://2010.igem.org/Team:Debrecen-Hungary/protocols/_Enzymatic_activity_with_Victor_machine Read more...]<br />
<br />
[[Team:Debrecen-Hungary/protocols#Notebook|[TOP]]]<br />
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==Online References At Openwetware==<br />
<br />
[https://2010.igem.org/Team:Debrecen-Hungary/protocols/makinglb ''Main article - Making Lurea's Broth'']<br />
<br />
[http://www.openwetware.org/wiki/IGEM:University_of_Debrecen:Transformation_of_competent_cells ''Main article - Transformation of competent cells'']<br />
<br />
[http://openwetware.org/wiki/Editing_IGEM:University_of_Debrecen:_Mini_Prep ''Main article - Mini prep'']<br />
<br />
[http://openwetware.org/wiki/IGEM:University_of_Debrecen:Restriction_biobrick_parts#Notes ''Main article - Restriction enzyme digestion'']<br />
<br />
[http://www.openwetware.org/wiki/IGEM:University_of_Debrecen:PCR_purification_from_Agarose_Gel ''Main article - PCR product purification / Gel purification''] <br><br />
<br />
[http://www.openwetware.org/wiki/IGEM:University_of_Debrecen:_Polymerase_Chain_Reaction_%28PCR%29 ''Main article - PCR''] <br><br />
<br />
[http://openwetware.org/wiki/IGEM:University_of_Debrecen:Gel_electrophoresis ''Main article - Gel electrophoresis'']<br />
<br />
[http://www.openwetware.org/wiki/IGEM:University_of_Debrecen:Cell_Passaging ''Main article - Cell passaging'']<br />
<br />
[http://openwetware.org/wiki/IGEM:University_of_Debrecen:_Media_PEI_Preparation ''Main article - Media PEI preparation'']<br />
<br />
[http://www.openwetware.org/wiki/IGEM:University_of_Debrecen:_Transfection ''Main article - Transfection'']<br />
<br />
[http://openwetware.org/wiki/IGEM:University_of_Debrecen:Ligand_Treatment ''Main article - Ligand treatment''] <br />
<br />
[http://openwetware.org/wiki/IGEM:University_of_Debrecen:_Enzymatic_activity_with_Victor_machine ''Main article - Measuring Luciferase activity with the Victor plate reader'']<br />
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<div></div>Liorhttp://2010.igem.org/Team:Debrecen-Hungary/galleryTeam:Debrecen-Hungary/gallery2010-10-27T10:25:54Z<p>Lior: </p>
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</div></div>Liorhttp://2010.igem.org/File:Igemgroup1.jpgFile:Igemgroup1.jpg2010-10-27T10:25:07Z<p>Lior: </p>
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<div></div>Liorhttp://2010.igem.org/Team:Debrecen-Hungary/galleryTeam:Debrecen-Hungary/gallery2010-10-27T10:22:39Z<p>Lior: </p>
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a:visited {color:#ede8e2;}<br />
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/* slides styling */<br />
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</div></div>Liorhttp://2010.igem.org/Team:Debrecen-Hungary/galleryTeam:Debrecen-Hungary/gallery2010-10-27T10:21:32Z<p>Lior: </p>
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<div>{{Template:Debrecen-Hungary_secret}}<br />
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<li><a href="https://static.igem.org/mediawiki/2010/6/67/0002.JPG"><img src="https://static.igem.org/mediawiki/2010/6/67/0002.JPG" alt="" title="" /></a></li><br />
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<li><a href="https://static.igem.org/mediawiki/2010/9/91/00018.jpg"><img src="https://static.igem.org/mediawiki/2010/0/04/00017.jpg" alt="" title="" /></a></li><br />
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<li><a href="https://static.igem.org/mediawiki/2010/4/48/00022.jpg"><img src="https://static.igem.org/mediawiki/2010/4/48/00022.jpg" alt="" title="" /></a></li><br />
<li><a href="https://static.igem.org/mediawiki/2010/c/c6/00023.jpg"><img src="https://static.igem.org/mediawiki/2010/c/c6/00023.jpg" alt="" title="" /></a></li><br />
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<br><br><br><br />
<center><br />
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__NOTOC__<br />
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</div></div>Liorhttp://2010.igem.org/Template:Debrecen-Hungary_secretTemplate:Debrecen-Hungary secret2010-10-27T10:08:02Z<p>Lior: </p>
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<div><html><br />
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Breaks O9.<br />
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Hidding it (* html) from non-IE browsers breaks IE7! <br />
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}<br />
div.MenuBar ul li:hover ul.DropDownMenu li:hover ul.SideMenu li a,<br />
div.MenuBar ul li a:hover ul.DropDownMenu li a:hover ul.SideMenu li a {<br />
background-color: Menu; /* Side menu unselected items.<br />
Sets the side menu "effective background" color. */<br />
color: MenuText;<br />
}<br />
div.MenuBar ul li:hover ul.DropDownMenu li:hover ul.SideMenu li a:hover,<br />
div.MenuBar ul li a:hover ul.DropDownMenu li a:hover ul.SideMenu li a:hover {<br />
background-color: Highlight; /* Side menu selected item. */<br />
color: HighlightText; <br />
}<br />
/*-----------------------------------------------------------------------------------------------*/<br />
/*Script-Free 3-Level Menu 1.2 Tailor<br />
www.CesarDaniel.info<br />
/*-------------------------------------------------------------------------------------- General */<br />
body {<br />
background: white;<br />
color: black;<br />
margin: 0;<br />
border: 0;<br />
padding: 0;<br />
}<br />
<br />
<br />
div.MenuBar {<br />
font: 13px arial, helvetica, sans-serif;<br />
}<br />
div.MenuBar ul {<br />
font: 13px arial, helvetica, sans-serif; /* Required for IE55. */<br />
}<br />
/*--------------------------------------------------------------------------------------- Colors */<br />
div.MenuBar {<br />
background-color: black; /* Selected item. */<br />
color: #FFFFFF;<br />
border-bottom: 1px solid ButtonShadow;<br />
}<br />
div.MenuBar a,<br />
div.MenuBar ul li:hover ul.DropDownMenu li a,<br />
div.MenuBar ul li a:hover ul.DropDownMenu li a,<br />
div.MenuBar ul li:hover ul.DropDownMenu li:hover ul.SideMenu li a,<br />
div.MenuBar ul li a:hover ul.DropDownMenu li a:hover ul.SideMenu li a {<br />
background-color: black; /* Selected item. */<br />
color: #FFFFFF;<br />
}<br />
div.MenuBar ul li:hover a,<br />
div.MenuBar ul li a:hover,<br />
div.MenuBar ul li:hover ul.DropDownMenu li:hover a,<br />
div.MenuBar ul li a:hover ul.DropDownMenu li a:hover,<br />
div.MenuBar ul li:hover ul.DropDownMenu li:hover ul.SideMenu li a:hover,<br />
div.MenuBar ul li a:hover ul.DropDownMenu li a:hover ul.SideMenu li a:hover {<br />
background-color: #00b0e6; /* Selected item. */<br />
color: #FFFFFF;<br />
}<br />
div.MenuBar ul li:hover ul.DropDownMenu,<br />
div.MenuBar ul li a:hover ul.DropDownMenu,<br />
div.MenuBar ul li:hover ul.DropDownMenu li:hover ul.SideMenu,<br />
div.MenuBar ul li a:hover ul.DropDownMenu li a:hover ul.SideMenu {<br />
background-color: ButtonShadow; /* Sets the drop-down and side menus "effective border" color. */<br />
}<br />
/*--------------------------------------------------------------------------------------- Widths */<br />
/*<br />
<br />
/*<br />
Menu Bar 1<br />
Drop-Down Menu #2<br />
*/<br />
div.MenuBar#navi ul li:hover ul.DropDownMenu#MB1-DDM4,<br />
div.MenuBar#navi ul li a:hover ul.DropDownMenu#MB1-DDM4,<br />
div.MenuBar#navi ul li:hover ul.DropDownMenu#MB1-DDM4 li a,<br />
div.MenuBar#navi ul li a:hover ul.DropDownMenu#MB1-DDM4 li a {<br />
width: 11em; /* Drop-down menu width. */<br />
}<br />
div.MenuBar#navi ul li:hover ul.DropDownMenu#MB1-DDM5,<br />
div.MenuBar#navi ul li a:hover ul.DropDownMenu#MB1-DDM5,<br />
div.MenuBar#navi ul li:hover ul.DropDownMenu#MB1-DDM5 li a,<br />
div.MenuBar#navi ul li a:hover ul.DropDownMenu#MB1-DDM5 li a {<br />
width: 12em; /* Drop-down menu width. */<br />
}<br />
<br />
/*...............................................................................................*/<br />
/*<br />
Menu Bar 1<br />
Drop-Down Menu #2<br />
Side Menu #1<br />
*/<br />
div.MenuBar#navi ul li:hover ul.DropDownMenu li:hover ul.SideMenu#MB1-DDM2-SM1,<br />
div.MenuBar#navi ul li a:hover ul.DropDownMenu li a:hover ul.SideMenu#MB1-DDM2-SM1 {<br />
left: 15.5em !important; /* Places the side menu to the right of the drop-down menu.<br />
Keep it sync with the drop-down menu width. */<br />
}<br />
div.MenuBar#navi ul li:hover ul.DropDownMenu li:hover ul.SideMenu#MB1-DDM2-SM1,<br />
div.MenuBar#navi ul li a:hover ul.DropDownMenu li a:hover ul.SideMenu#MB1-DDM2-SM1,<br />
div.MenuBar#navi ul li:hover ul.DropDownMenu li:hover ul.SideMenu#MB1-DDM2-SM1 li a,<br />
div.MenuBar#navi ul li a:hover ul.DropDownMenu li a:hover ul.SideMenu#MB1-DDM2-SM1 li a {<br />
width: 10em; /* Side menu width. */<br />
}<br />
/*...............................................................................................*/<br />
/*<br />
Menu Bar 1<br />
Drop-Down Menu #2<br />
Side Menu #2<br />
*/<br />
div.MenuBar#navi ul li:hover ul.DropDownMenu li:hover ul.SideMenu#MB1-DDM2-SM2,<br />
div.MenuBar#navi ul li a:hover ul.DropDownMenu li a:hover ul.SideMenu#MB1-DDM2-SM2 {<br />
left: 15.5em !important; /* Places the side menu to the right of the drop-down menu.<br />
Keep it sync with the drop-down menu width. */<br />
}<br />
div.MenuBar#navi ul li:hover ul.DropDownMenu li:hover ul.SideMenu#MB1-DDM2-SM2,<br />
div.MenuBar#navi ul li a:hover ul.DropDownMenu li a:hover ul.SideMenu#MB1-DDM2-SM2,<br />
div.MenuBar#navi ul li:hover ul.DropDownMenu li:hover ul.SideMenu#MB1-DDM2-SM2 li a,<br />
div.MenuBar#navi ul li a:hover ul.DropDownMenu li a:hover ul.SideMenu#MB1-DDM2-SM2 li a {<br />
width: 10em; /* Side menu width. */<br />
}<br />
/*...............................................................................................*/<br />
/*<br />
Menu Bar 1<br />
Drop-Down Menu #2<br />
Side Menu #3<br />
*/<br />
div.MenuBar#navi ul li:hover ul.DropDownMenu li:hover ul.SideMenu#MB1-DDM2-SM3,<br />
div.MenuBar#navi ul li a:hover ul.DropDownMenu li a:hover ul.SideMenu#MB1-DDM2-SM3 {<br />
left: 15.5em !important; /* Places the side menu to the right of the drop-down menu.<br />
Keep it sync with the drop-down menu width. */<br />
}<br />
div.MenuBar#navi ul li:hover ul.DropDownMenu li:hover ul.SideMenu#MB1-DDM2-SM3,<br />
div.MenuBar#navi ul li a:hover ul.DropDownMenu li a:hover ul.SideMenu#MB1-DDM2-SM3,<br />
div.MenuBar#navi ul li:hover ul.DropDownMenu li:hover ul.SideMenu#MB1-DDM2-SM3 li a,<br />
div.MenuBar#navi ul li a:hover ul.DropDownMenu li a:hover ul.SideMenu#MB1-DDM2-SM3 li a {<br />
width: 10em; /* Side menu width. */<br />
}<br />
/*...............................................................................................*/<br />
<br />
</style><br />
<br />
<br />
<br />
<br />
<br />
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</center><br />
<!---unibanner><br />
<div id="header"><a href="https://2010.igem.org/Team:Debrecen-Hungary"><img src="http://imgur.com/6rsGo.jpg" width="940" height="340" alt="Team Debrecen" /></a></div><br />
<br />
<br />
---><br />
<div class='MenuBar' id="navi"><br />
<ul><br />
<br />
<li><br />
<a href="https://2010.igem.org/Team:Debrecen-Hungary/Team" style="color: white"><img src="http://imgur.com/R3OOO.gif" /> Team<!--[if gt IE 6]><!--></a><!--<![endif]--><br />
<!--[if lt IE 7]><table border="0" cellpadding="0" cellspacing="0"><tr><td><![endif]--><br />
<ul class="DropDownMenu" id="MB1-DDM6"><br />
<li><a href="https://2010.igem.org/Team:Debrecen-Hungary/meetteam"><span><span>Meet the Team</span></span></a></li><br />
<li><a href="https://2010.igem.org/Team:Debrecen-Hungary/gallery"><span><span>Gallery</span></span></a></li><br />
<li><a href="https://2010.igem.org/Team:Debrecen-Hungary/Team#about"><span><span>About The team</span></span></a></li><br />
<br />
<br />
<li><a href="https://2010.igem.org/Team:Debrecen-Hungary/Team#Lab"><span><span>About The Lab</span></span></a></li><br />
<li><a href="https://2010.igem.org/Team:Debrecen-Hungary/Team#deoec"><span><span>About The University</span></span></a></li><br />
<li><a href="https://2010.igem.org/Team:Debrecen-Hungary/Team#debrecen"><span><span>About Debrecen</span></span></a></li><br />
<li><a href="https://2010.igem.org/Team:Debrecen-Hungary/Team#cube"><span><span>About Rubiks's Cube</span></span></a></li><br />
<br />
<br />
</ul><br />
<!--[if lte IE 6]></td></tr></table></a><![endif]--><br />
</li><br />
<li><br />
<a href="https://2010.igem.org/Team:Debrecen-Hungary/project" style="color: white"><img src="http://imgur.com/R3OOO.gif" /> Project<!--[if gt IE 6]><!--></a><!--<![endif]--><br />
<!--[if lt IE 7]><table border="0" cellpadding="0" cellspacing="0"><tr><td><![endif]--><br />
<ul class="DropDownMenu" id="MB1-DDM1"><br />
<li><a href="https://2010.igem.org/Team:Debrecen-Hungary/project#abs"><span><span>Abstract</span></span></a></li><br />
<li><a href="https://2010.igem.org/Team:Debrecen-Hungary/project#intro"><span><span>Introduction</span></span></a><br />
<li><a href="https://2010.igem.org/Team:Debrecen-Hungary/project#Results"><span><span>Results</span></span></a></li><br />
<li><a href="https://2010.igem.org/Team:Debrecen-Hungary/project#Plans"><span><span>Plans, Possibilities</span></span></a></li><br />
<li><a href="https://2010.igem.org/Team:Debrecen-Hungary/project#BBF_RFC_64_:_Building_Protein_Domain_Based_Composite_Biobricks_for_Mammalian_Expression_Systems"><span><span>RFC 64 suggestion</span></span></a></li><br />
<li><a href="https://2010.igem.org/Team:Debrecen-Hungary/indicontibi"><span><span>Members Contribution</span></span></a></li><br />
<br />
</ul><br />
<!--[if lte IE 6]></td></tr></table></a><![endif]--><br />
</li><br />
<br />
<li><br />
<a href="https://2010.igem.org/Team:Debrecen-Hungary/parts" style="color: white"><img src="http://imgur.com/R3OOO.gif" /> Parts<!--[if gt IE 6]><!--></a><!--<![endif]--><br />
<!--[if lt IE 7]><table border="0" cellpadding="0" cellspacing="0"><tr><td><![endif]--><br />
<ul class="DropDownMenu" id="MB1-DDM2"><br />
<li><a href="https://2010.igem.org/Team:Debrecen-Hungary/parts#LBD"><span><span>LBD</span></span></a></li><br />
<li><a href="https://2010.igem.org/Team:Debrecen-Hungary/parts#DBD"><span><span>DBD</span></span></a></li><br />
<li><a href="https://2010.igem.org/Team:Debrecen-Hungary/parts#other"><span><span>Other basic parts</span></span></a></li><br />
<li><a href="https://2010.igem.org/Team:Debrecen-Hungary/parts#composite"><span><span>Composite Parts</span></span></a></li><br />
<li><a href="https://2010.igem.org/Team:Debrecen-Hungary/favorite"><span><span>Favorite Parts</span></span></a></li><br />
<br />
</ul><br />
<!--[if lte IE 6]></td></tr></table></a><![endif]--><br />
</li><br />
<li><br />
<a href="https://2010.igem.org/Team:Debrecen-Hungary/protocols" style="color: white"><img src="http://imgur.com/R3OOO.gif" /> Protocols<!--[if gt IE 6]><!--></a><!--<![endif]--><br />
<!--[if lt IE 7]><table border="0" cellpadding="0" cellspacing="0"><tr><td><![endif]--><br />
<ul class="DropDownMenu" id="MB1-DDM3"><br />
<br />
<li><a href="https://2010.igem.org/Team:Debrecen-Hungary/protocols#Contents"><span><span>Cloning Team</span></span></a></li><br />
<li><a href="https://2010.igem.org/Team:Debrecen-Hungary/protocols#Mini_Prep"><span><span>Molecular work team</span></span></a></li><br />
<li><a href="https://2010.igem.org/Team:Debrecen-Hungary/protocols#Gel_electrophoresis"><span><span>Tissue culture team</span></span></a></li><br />
<li><a href="https://2010.igem.org/Team:Debrecen-Hungary/protocols#Measuring_Luciferase_activity_with_the_Victor_plate_reader"><span><span>Luciferase Team</span></span></a></li><br />
</ul><br />
<!--[if lte IE 6]></td></tr></table></a><![endif]--><br />
</li><br />
<br />
<br />
<br />
<br />
<li><br />
<a href="https://2010.igem.org/Team:Debrecen-Hungary/sponsors" style="color: white"><img src="http://imgur.com/R3OOO.gif" /> Sponsors<!--[if gt IE 6]><!--></a><!--<![endif]--><br />
<!--[if lt IE 7]><table border="0" cellpadding="0" cellspacing="0"><tr><td><![endif]--><br />
<!--here--><br />
</li><br />
<br />
<li><br />
<a style="color: white"><img src="http://imgur.com/R3OOO.gif" /> Tools<!--[if gt IE 6]><!--></a><!--<![endif]--><br />
<!--[if lt IE 7]><table border="0" cellpadding="0" cellspacing="0"><tr><td><![endif]--><br />
<!--here--><br />
<br />
<br />
<ul class="DropDownMenu" id="MB1-DDM3"><br />
<li><a href="https://2010.igem.org/Team:Debrecen-Hungary/sitemap"><span><span>Site Map</span></span></a></li><br />
<li><a href="https://2010.igem.org/Team:Debrecen-Hungary/minimals"><span><span>The Minimals</span></span></a></li><br />
<li><a href="https://2010.igem.org/Team:Debrecen-Hungary/safety"><span><span>Safety</span></span></a></li><br />
<li><a href="https://2010.igem.org/Team:Debrecen-Hungary/Team_meetings"><span><span>Teem Meetings</span></span></a></li><br />
<li><a href="https://2010.igem.org/Team:Debrecen-Hungary/links"><span><span>Links</span></span></a></li><br />
</ul><br />
</li><br />
<br />
<li><br />
<a style="color: white"><img src="http://imgur.com/R3OOO.gif" /> Side Projects<!--[if gt IE 6]><!--></a><!--<![endif]--><br />
<!--[if lt IE 7]><table border="0" cellpadding="0" cellspacing="0"><tr><td><![endif]--><br />
<!--here--><br />
<br />
<ul class="DropDownMenu" id="MB1-DDM3"><br />
<li><a href="https://2010.igem.org/Team:Debrecen-Hungary/arsenic"><span><span>Arsenic Project</span></span></a></li><br />
<br />
<li><a href="https://2010.igem.org/Team:Debrecen-Hungary/AKG_team"><span><span>AKG team</span></span></a></li><br />
<li><a href="https://2010.igem.org/Team:Debrecen-Hungary/video_project"><span><span>Video Project</span></span></a></li><br />
<li><a href="https://2010.igem.org/Team:Debrecen-Hungary/on_the_media"><span><span>On The Media</span></span></a></li><br />
<br />
<br />
</li><br />
<br />
</ul><br />
<br />
</div><br />
<br />
<style><br />
<br />
body {background: url(http://imgur.com/3Yr12.jpg);}<br />
<br />
</style><br />
<br />
<br />
<br />
<br />
</html></div>Liorhttp://2010.igem.org/Template:Debrecen-Hungary_secretTemplate:Debrecen-Hungary secret2010-10-27T10:00:52Z<p>Lior: </p>
<hr />
<div><html><br />
<br />
<body id="template:debrecen-hungary_secret" ></body></html><br />
{|<br />
|-valign="top" border="0" style="margin-left: 0px;" style="margin-right: 0px;"<br />
|width="1100px" style="padding: 0 0px 0px 0px; background-color:#ede8e2"|<br />
<br />
<html><br />
<br />
<style><br />
<br />
<br />
h1.firstHeading { display: none; }<br />
<br />
p {text-align: justify;}<br />
<br />
a:link { color: #00b0e6; text-decoration: none} <br />
a:visited { color:#00b0e6; text-decoration: none}<br />
a:hover { color:#f29400; text-decoration: none}<br />
a:active { color:#f29400; text-decoration: none}<br />
<br />
#bodyContent { padding: 5px auto; width: 968px; margin: auto; clear: none; }<br />
<br />
table#team_members { text-align: justify; border: 0; }<br />
table#team_members h2, table#team_members h3 { clear: both; }<br />
<br />
<br />
/*-----------------------------------------------------------------------------------------------*/<br />
div.MenuBar ul li ul.DropDownMenu {<br />
display: none; /* Hides all drop-down menus. */<br />
<br />
}<br />
/*<br />
li:hover works in IE7 and FF2.<br />
a:hover works in IE6 and FF2.<br />
a:hover breaks li:hover in FF2.<br />
*/<br />
div.MenuBar ul li:hover ul.DropDownMenu li ul.SideMenu,<br />
div.MenuBar ul li a:hover ul.DropDownMenu li a ul.SideMenu {<br />
display: none; /* Hides all side menus. */<br />
}<br />
/*------------------------------------------------------------------------------------- Menu Bar */<br />
div.MenuBar {<br />
font: arial, helvetica, sans-serif ;<br />
height: 30px;<br />
width: 960px;<br />
/*width: 100%*/<br />
margin: 0;<br />
border-top: 0;<br />
border-right: 0;<br />
border-left: 0;<br />
padding: 0;<br />
background: black;<br />
<br />
}<br />
div.MenuBar ul {<br />
font: arial, helvetica, sans-serif;<br />
text-align: center; <br />
list-style-type: none;<br />
margin: 0.5em auto;<br />
border: 0;<br />
padding: 0;<br />
background: black;<br />
}<br />
div.MenuBar ul li {<br />
font: arial, helvetica, sans-serif;<br />
display: block;<br />
padding: 0;<br />
margin: 0;<br />
font-size: 1 em;<br />
float: left;<br />
background: black;<br />
text-align: left;<br />
width: 130px;<br />
position: relative; /* Sets the positioning context for each drop-down menu. */<br />
}<br />
<br />
div.MenuBar ul li a {<br />
font: arial, helvetica, sans-serif;<br />
display: block;<br />
background: black;<br />
height: 22px; /* Keep height + padding-top + padding-bottom sync with the menu bar height. */<br />
color: #ffffff;<br />
padding-top: 4px;<br />
padding-bottom: 4px;<br />
padding-left: 1em; /* Sets the left space between top-level items. */<br />
padding-right: 1em; /* Sets the right space between top-level items. */<br />
text-decoration: none;<br />
}<br />
<br />
/*------------------------------------------------------------------------------ Drop-Down Menus */<br />
div.MenuBar ul li:hover ul.DropDownMenu,<br />
div.MenuBar ul li a:hover ul.DropDownMenu {<br />
display: block;<br />
width: 12.5em; /* Drop-down menu width.<br />
Use MenuTailor.css to customize. */<br />
height: 0.5em;<br />
padding: 1px; /* Sets the drop-down menu "effective border" width. */<br />
position: absolute; <br />
top: 23px; /* Places the drop-down menu under the menu bar.<br />
Keep it sync with the menu bar height. */<br />
left: 0; /* Aligns the drop-down menu to its top-level item. */<br />
background-color: black; /* Selected item. */<br />
color: #FFFFFF;<br />
<br />
}<br />
div.MenuBar ul li:hover ul.DropDownMenu li a,<br />
div.MenuBar ul li a:hover ul.DropDownMenu li a {<br />
width: 12.5em; /* Keep it sync with the drop-down menu width.<br />
Use MenuTailor.css to customize. */<br />
height: 1.5em;<br />
padding-left: 0;<br />
padding-right: 0;<br />
background-color: black; /* Selected item. */<br />
color: #FFFFFF;<br />
}<br />
ul.DropDownMenu li a span {<br />
display: block;<br />
padding-left: 0.75em; /* Sets the left space of each drop-down menu item. */<br />
padding-right: 0.25em; /* Sets the right space of each drop-down menu item. */<br />
text-align: right; /* Aligns the >> symbol to the right. */<br />
}<br />
ul.DropDownMenu li a span span {<br />
float: left; /* Aligns the text (back) to the left. */<br />
font: 12px arial, helvetica, sans-serif; /* Required for IE55. */<br />
height: 20px;<br />
color: #FFFFFF;<br />
}<br />
/*----------------------------------------------------------------------------------- Side Menus */<br />
div.MenuBar ul li:hover ul.DropDownMenu li:hover ul.SideMenu,<br />
div.MenuBar ul li a:hover ul.DropDownMenu li a:hover ul.SideMenu {<br />
display: block;<br />
width: 11em; /* Side menu width.<br />
Use MenuTailor.css to customize. */<br />
padding: 1px; /* Sets the side menu "effective border" width. */<br />
position: absolute;<br />
top: -1px; /* Aligns the side menu to its drop-down menu item.<br />
Keep it sync with the side menu "effective border" width. */<br />
left: 13em; /* Places the side menu to the right of the drop-down menu.<br />
Keep it sync with the drop-down menu width.<br />
Use MenuTailor.css to customize. */<br />
}<br />
div.MenuBar ul li:hover ul.DropDownMenu li:hover ul.SideMenu li a,<br />
div.MenuBar ul li a:hover ul.DropDownMenu li a:hover ul.SideMenu li a {<br />
width: 11em; /* Keep it sync with the side menu width.<br />
Use MenuTailor.css to customize. */<br />
font: 12px arial, helvetica, sans-serif; /* Required for IE55. */<br />
left: 13em; /* Places the side menu to the right of the drop-down menu.<br />
Keep it sync with the drop-down menu width.<br />
Use MenuTailor.css to customize. */<br />
}<br />
div.MenuBar ul li ul.DropDownMenu li ul.SideMenu li a span {<br />
padding-left: 1.5em; /* Sets the left space of each side menu item. */<br />
padding-right: 0.5em; /* Sets the right space of each side menu item. */<br />
text-align: left;<br />
font: 12px arial, helvetica, sans-serif; /* Required for IE55. */<br />
left: 13em; /* Places the side menu to the right of the drop-down menu.<br />
Keep it sync with the drop-down menu width.<br />
Use MenuTailor.css to customize. */<br />
}<br />
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</html></div>Liorhttp://2010.igem.org/Team:Debrecen-Hungary/protocols/_TransfectionTeam:Debrecen-Hungary/protocols/ Transfection2010-10-27T09:51:24Z<p>Lior: </p>
<hr />
<div>{{Template:Debrecen-Hungary_secret}}<br />
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<br />
===FuGENE 6-nuclear receptor transfection of COS1 cells===<br />
<br />
==Scientific Background==<br />
<br />
FuGENE 6 transfection reagent is a multi-component lipid reagent that forms a complex with the DNA, then transports it into animal cells. FuGENE 6 transfection is used as standard method in many different laboratories due to its simple methodology, low cytotoxicity, and ability to provide high transfection efficiency even in the presence of serum. <br />
<br />
==Overview==<br />
<br />
The transfection is divided into two main steps:<br />
1. plating COS1 cells into glass cell culture vessel<br />
2. the transfection itself<br />
<br />
<h5>1. Plating COS1 cells into glass cell culture vessel:</h5><br />
The aim is to get cells of 50-80% confluency in the chosen vessel for the day of transfection. <br />
<br />
Required materials:<br><br />
<br />
- COS1 cells in a T75 flask (regularly passaged, proliferating well - best in the log-growth phase)<br><br />
- Trypsin/EDTA solution<br><br />
- 1 x PBS solution<br><br />
- 10% FBS containing DMEM medium<br><br />
- LAB-TEK glass chamber slide with 8 chambers<Br><br />
- 15 ml centrifuge tube, tube holders<br><br />
- Bürker chamber, pipettor, serological pipettes, pipettes, pipet tips, Pasteur pipettes<br><br />
- sterile laminar air flow box, 37°C incubator, 37°C waterbath<br><br />
- 70% ethanol, kimwipes<br><br />
<br />
<h5>Steps:</h5><br><br />
1. Prepare the sterile box: Open the sterile laminar box (Hood), turn on the ventillator and wait for 15 minutes<br />
to reach the optimal level of sterility (0,45 um filter). PUT ON YOUR GLOVES, wipe the box with 70% alcohol.<br><br><br><br />
<br />
2. Prewarm the DMEM medium, Trypsin/EDTA and the 1x PBS in the 37°C waterbath (10-15 minutes)<br><br><br><br />
<br />
3. Take the DMEM, Trypsin-EDTA and PBS and squirt the tubes and bottles with alcohol beforeyou put them in the sterile box. Put the pipettor and the tube holder into the box <br><br />
(after you sprayed them down with 70% ethanol), and load the serlogical pipettes directly,without spraying down into the box.<br><br><br><br />
<br />
4. Spray hands with ethanol. Remove the flask from the incubator and quickly place in hood. Fire-sterilize the neck of the<br> flask. (Do not spray flasks with ethanol).<br><br><br><br />
<br />
5. Attach a Pasteur pipette to vacuum,turn on vacuum system by opening vacuum valve in hood. You should fire-sterilize the end of the pipette,after this step do not touch anything outside the flask. Aspirate the used medium from the cells by touching the bottom-side corner of the flask with the Pasteur-pipette.<br><br><br><br />
<br />
6. Washing step:Add 2-3 mL of 1x PBS to flask by using pipettor and a serological pipette (Release the PBS onto the side of the flask, do not push the solution out directly onto the cells because they can come up easily). <br />
Lightly swirl PBS on base of the flask. Aspirate PBS from flasks by using a Pasteur pipette and vacuum.<br><br><br><br />
<br />
7. Add 2 mL trypsin-EDTA to Flask. You can release the solution directly onto the cells, <br />
from now it does not matter if they come up. Lightly swish trypsin.<br><br><Br><br />
<br />
8. Place flask in 37°C incubator until detached (3-5 minutes for COS1 cells, depending on the temperature of the Trypsin- opt. temp: 37°C)<br><Br><Br><br />
<br />
9. Remove cells from incubator. Tap side of the flask on hard surface of your hand. Repeat several times. Visually check to ensure lumps of cells are dispersed.<br><br><br><br />
<br />
10. Check cells under phase-contrast microscope to confirm that cells are detached from the surface.<br><br><Br><br />
<br />
11. Put the falsk back to the sterile box, add 4 ml of 10% FBS containing DMEM medium to dilute trypsin (you can change the dilution level depending on the cell number, in order to be able to count the cells easier). Medium contains antitrypsin.(Note: The liquid suspension now contains the cells.) <br><br><br><br />
<br />
12. Carefully resuspend cells by using pipettor and serological pipettes. You can repeat this step until you get individual floating cells (microscope check needed).Put the cells into a 50 ml tube, for easier handling.<br><br><br><br />
<br />
13. Prepare the Bürker chamber and do a cell counting:<br><br><br><br />
<br />
<h5>- Cell counting in a Bürker chamber:</h5><br />
The Bürker chamber is the most commonly used tool for counting cells. Now comes a short introduction about how to use a Bürker chamber:<br><br><br />
<br />
<br />
* The Bürker chamber from outside: <br><br />
<html><br />
<img src="https://static.igem.org/mediawiki/2010/b/bc/Burker1.jpg" alirgn="left"><br />
</html><br><br><br />
<br />
<h5>* How to use the Bürker chamber:</h5><br><br><br />
<br />
1) At first, clean the chamber with alcohol and water, put the thin glass slide onto the thicker slide,<br><br />
compress them together by using the metal screw.<br><br><br />
2) Resuspend the cell suspension (for even distribution) in which you want to do the cell counting.<br> <br><br />
3) Take 10 ul from the cell suspension (using sterile tip), <br><br><br />
inject into the chamber(marked with arrow) by touching the border of the two glasses with the pipet tip.<br><br><br />
4) Place the chamber under the phase-contrast microscope and try to find the lines.<br><br><br />
<br />
- count inside 3 big squares (an example is on the next picture): <br><br><br />
<br />
<html><br />
<img src="https://static.igem.org/mediawiki/2010/c/c3/Burker2.jpg" alirgn="left"><br />
</html><br><br><br />
<br />
<br />
then we take the average of the 3 big squares, and it will give us the number of the cells in 0,1 ul<br><Br><br />
we take it 10 in the factor of 4 times and it will give us the cell number in 1 ml cell suspension.<br><br><br />
<br />
NOTE: Don't forget that the cell suspenson in the chamber is now not sterile,<br><br />
don't put it back into the cell solution. Clean the chamber with alcohol and water.<br><br />
<br />
<br />
1. To reach the appropriate confluency the day after plating, we put 50.000 cells into each well.<br><br />
For an 8 well chamber, if we calculate with 12 wells (because the volume loss), we put into a 15 ml centrifuge tube:<br><br />
- 50.000 x 12 = 600.000 cells [ in milliliter: counted cell number in 1 ml / 600.000 cells ]<br><br />
- we fill the cell suspension up to 8 x 300 ul = 2, 5 ml with 10% FBS DMEM<br><br />
(total volume of the wells are 300 ul)<Br><br><br />
2. With a 1 ml pipette put 300 ul from this suspension into each well. Sometimes invert the cell suspension containing tube <br>(the cells decent). After you finished the plate, swirl it circularly.<br><Br><br />
3. Incubate the cells for 1 day at 37°C, 5% CO2.<br><br><br />
<br />
<br />
<br />
<h5>2. FuGENE 6 transfection:</h5><Br><br />
The goal is to introduce foreign plasmid DNA into the plated COS1 cells 24 hours after the plating.<Br><Br><br />
<br />
Materials required:<Br><br />
- Plated cells in a LAB-TEK 8 well glass chamber slide ( with 50-80% confluency)<br><br />
- FBS/antibiotics/other additives Free DMEM medium<br><br />
- Sterile FuGENE 6 transfection reagent in a tightly capped glass vial<br><br />
- Plasmids on ice, in known concentrations: Beta-Gal (normalizer plasmid), Luciferase (tracer), Nuclear receptor, VDR- as <br>negative control, the plasmids are solved in sterile TE-buffer<br><br />
- 1,5 ml sterile Eppendorf tube, pipettes and tips<Br><br />
- Sterile laminar air flow box, 37°C incubator, 37°C waterbath<br><br />
- 70% ethanol squirt bottle, kimwipes<br><Br><br />
<br />
<br />
<h5>Steps:</h5><Br><br />
1. Prepare the sterile box: Open the sterile laminar box (Hood), turn on the ventillator and wait for 15 minutes to reach the optimal level of sterility (0,45 um filter). PUT ON YOUR GLOVES, spray down the base of the box with 70% ethanol, and wipe down with kimwipes.<br><br><br />
<br />
2. Spray down your hands, the DMEM medium, the glass Reagent vial and the pipettes and tips with 70% ethanol. Put these and the plasmids and the eppendorf tube into the sterile box.<br><Br><br />
<br />
3. Transfection mix reconstitution:<br><br />
<br />
&nbsp;&nbsp;3.1 Invert the room temp. FuGEGE 6 Transfection Reagent glass vial 2-3 times to distribute the components.<br><br />
&nbsp;&nbsp;3.2 Dilute the FuGENE reagent with Serume/antibiotics/other additives free DMEM medium – the order and manner of &nbsp;&nbsp;addition is critical:<Br><br />
&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Label a 1,5 ml eppendorf tube.<Br><br />
&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Pipet 75 ul FBS/antibiotics/other additives Free DMEM into the eppendorf tube.<Br><br />
&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Pipet 6 ul FuGENE 6 Transfection Reagent directly into the medium, without allowing contact between the plastic &nbsp;&nbsp;&nbsp;wall and the undiluted reagent.<br><br />
&nbsp;&nbsp;3.3 Vortex the mix for 1 second.<br><br />
&nbsp;&nbsp;3.4 Incubate the mix for 5 minutes at room temperature.<br><br />
&nbsp;&nbsp;3.5 Add 500 ng from each of the three plasmids (receptor, Luciferase, beta – Gal.) into the diluted FuGENE 6 &nbsp;&nbsp;transfection reagent.<br><br />
&nbsp;&nbsp;3.6 Vortex the Transfection Reagent:Plasmid mixture for 1 second.<Br><br />
&nbsp;&nbsp;3.7 Incubate the mixture for 20 minutes at room temperature.<br><br><br />
<br />
4. Remove glass chamber slide with plated cells from the incubator, place in the sterile box without spraying down with ethanol.<br><Br><br />
<br />
5. Note: We don’t need to remove the culturing medium (10% FBS containing DMEM) from the cells, it does not have any effect on <Br>the transfection efficiency.<br><br />
Add 9 ul Transfection mix in a dropwise manner to each well. Swirl the chamber slide to ensure distribution over the entire surface.<Br><Br><br />
<br />
6. Put on the cap of the slide chamber. Return the cells to the 37°C incubator until the assay for gene expression is to be<br> performed.<br><br />
Note: it is not necessary to remove and replace the transfection mixture-containing medium with fresh medium until the assay, only if you used FBS Free medium during the whole experiment (to avoid the cell starvation).<Br><br><br />
<br />
7. Clean up after yourself, place the FuGENE reagent to +2 - +8 °C and be sure if the cap is tightly turned on the Reagent.<Br><br><br />
<br />
<br />
<h5>NOTES:</h5><br><br />
- store the reagent at +2 - +8, with the lid very tightly closed, in the original glass vial.<Br><br />
- Do not allow the reagent to contact plastic walls (pipet directly into serum free medium) to keep the maximal biological activity.<Br><br />
- Do not use siliconized pipet tips and tubes.<Br><br />
- To prepare transfection complexes for larger experiments or parallel experiments, proportionally increase the quantity <br>according to the total surface area of the cell culture vessel being used. (ul FuGENE Reagent: ug DNA = 4:1, the used vessel in <Br>this case has a 79,21 cm2 surface area)<br><br />
<br />
<br />
<html><br />
<object style="height: 190px; width: 290px" ><param name="movie" value="http://www.youtube.com/v/r8DlyOCRJRs" ><param name="allowFullScreen" value="true" ><param name="allowScriptAccess" value="always" ><embed src="http://www.youtube.com/v/r8DlyOCRJRs" type="application/x-shockwave-flash" allowfullscreen="true" allowScriptAccess="always" width="290" height="190"></object><br />
</html><br />
&nbsp;&nbsp;<br />
<html><br />
<object style="height: 190px; width: 290px" ><param name="movie" value="http://www.youtube.com/v/IgRxD-Tf5Eo" ><param name="allowFullScreen" value="true" ><param name="allowScriptAccess" value="always" ><embed src="http://www.youtube.com/v/IgRxD-Tf5Eo" type="application/x-shockwave-flash" allowfullscreen="true" allowScriptAccess="always" width="290" height="190" ></object><br />
</html><br />
<Br><br><br />
==Notes & troubleshooting==<br />
==References==<br />
1. Horbinski C, Stachowiak MK, Higgins D, Finnegan SG. Polyethyleneimine-mediated transfection of cultured postmitotic neurons from rat sympathetic ganglia and adult human retina. .[http://www.biomedcentral.com/1471-2202/2/2 BMC Neurosci. 2001;2:2].<br />
<br />
2. Pollard H, Remy JS, Loussouarn G, Demolombe S, Behr JP, Escande D: Polyethylenimine but not cationic lipids promotes transgene delivery to the nucleus in mammalian cells.[http://www.jbc.org/content/273/13/7507.long J Biol Chem 1998], 273:7507-7511<br />
<br />
==Links==<br />
<br />
[http://www.youtube.com/user/debrecenigem2010#p/u/13/r8DlyOCRJRs Video I]<br />
<br />
[http://www.youtube.com/user/debrecenigem2010#p/u/12/IgRxD-Tf5Eo Video II]</div>Liorhttp://2010.igem.org/Team:Debrecen-Hungary/protocols/_TransfectionTeam:Debrecen-Hungary/protocols/ Transfection2010-10-27T09:46:49Z<p>Lior: </p>
<hr />
<div>{{Template:Debrecen-Hungary_secret}}<br />
<br />
===FuGENE 6-nuclear receptor transfection of COS1 cells===<br />
<br />
==Scientific Background==<br />
<br />
FuGENE 6 transfection reagent is a multi-component lipid reagent that forms a complex with the DNA, then transports it into animal cells. FuGENE 6 transfection is used as standard method in many different laboratories due to its simple methodology, low cytotoxicity, and ability to provide high transfection efficiency even in the presence of serum. <br />
<br />
==Overview==<br />
<br />
The transfection is divided into two main steps:<br />
1. plating COS1 cells into glass cell culture vessel<br />
2. the transfection itself<br />
<br />
<h5>1. Plating COS1 cells into glass cell culture vessel:</h5><br />
The aim is to get cells of 50-80% confluency in the chosen vessel for the day of transfection. <br />
<br />
Required materials:<br><br />
<br />
- COS1 cells in a T75 flask (regularly passaged, proliferating well - best in the log-growth phase)<br><br />
- Trypsin/EDTA solution<br><br />
- 1 x PBS solution<br><br />
- 10% FBS containing DMEM medium<br><br />
- LAB-TEK glass chamber slide with 8 chambers<Br><br />
- 15 ml centrifuge tube, tube holders<br><br />
- Bürker chamber, pipettor, serological pipettes, pipettes, pipet tips, Pasteur pipettes<br><br />
- sterile laminar air flow box, 37°C incubator, 37°C waterbath<br><br />
- 70% ethanol, kimwipes<br><br />
<br />
<h5>Steps:</h5><br><br />
1. Prepare the sterile box: Open the sterile laminar box (Hood), turn on the ventillator and wait for 15 minutes<br><br />
to reach the optimal level of sterility (0,45 um filter). PUT ON YOUR GLOVES, wipe the box with 70% alcohol.<br><br><br><br />
<br />
2. Prewarm the DMEM medium, Trypsin/EDTA and the 1x PBS in the 37°C waterbath (10-15 minutes)<br><br><br><br />
<br />
3. Take the DMEM, Trypsin-EDTA and PBS and squirt the tubes and bottles with alcohol before<br><br />
you put them in the sterile box. Put the pipettor and the tube holder into the box <br><br />
(after you sprayed them down with 70% ethanol), and load the serlogical pipettes directly, <br><br />
without spraying down into the box.<br><br><br><br />
<br />
4. Spray hands with ethanol. Remove the flask from the incubator and quickly place in hood. Fire-sterilize the neck of the<br> flask. (Do not spray flasks with ethanol).<br><br><br><br />
<br />
5. Attach a Pasteur pipette to vacuum,<br><br />
turn on vacuum system by opening vacuum valve in hood. You should fire-sterilize the end of the pipette,<br><br />
after this step do not touch anything outside the flask. Aspirate the used medium from the cells by touching<br><br />
the bottom-side corner of the flask with the Pasteur-pipette.<br><br><br><br />
<br />
6. Washing step:Add 2-3 mL of 1x PBS to flask by using pipettor and a serological pipette (Release the PBS onto the side<br> of the flask, do not push the solution out directly onto the cells because they can come up easily).<br> <br />
Lightly swirl PBS on base of the flask. Aspirate PBS from flasks by using a Pasteur pipette and vacuum.<br><br><br><br />
<br />
7. Add 2 mL trypsin-EDTA to Flask. You can release the solution directly onto the cells, <br><br />
from now it does not matter if they come up. Lightly swish trypsin.<br><br><Br><br />
<br />
8. Place flask in 37°C incubator until detached (3-5 minutes for COS1 cells, <br><br />
depending on the temperature of the Trypsin- opt. temp: 37°C)<br><Br><Br><br />
<br />
9. Remove cells from incubator. Tap side of the flask on hard surface of your hand. <br><br />
Repeat several times. Visually check to ensure lumps of cells are dispersed.<br><br><br><br />
<br />
10. Check cells under phase-contrast microscope to confirm that cells are detached from the surface.<br><br><Br><br />
<br />
11. Put the falsk back to the sterile box, add 4 ml of 10% FBS containing DMEM medium to dilute trypsin <br><br />
(you can change the dilution level depending on the cell number, in order to be able to count the cells easier). <br><br />
Medium contains antitrypsin.(Note: The liquid suspension now contains the cells.) <br><br><br><br />
<br />
12. Carefully resuspend cells by using pipettor and serological pipettes. <br><br />
You can repeat this step until you get individual floating cells (microscope check needed). <br><br />
Put the cells into a 50 ml tube, for easier handling.<br><br><br><br />
<br />
13. Prepare the Bürker chamber and do a cell counting:<br><br><br><br />
<br />
<h5>- Cell counting in a Bürker chamber:</h5><br />
The Bürker chamber is the most commonly used tool for counting cells. Now comes a short introduction about how to use a Bürker chamber:<br><br><br />
<br />
<br />
* The Bürker chamber from outside: <br><br />
<html><br />
<img src="https://static.igem.org/mediawiki/2010/b/bc/Burker1.jpg" alirgn="left"><br />
</html><br><br><br />
<br />
<h5>* How to use the Bürker chamber:</h5><br><br><br />
<br />
1) At first, clean the chamber with alcohol and water, put the thin glass slide onto the thicker slide,<br><br />
compress them together by using the metal screw.<br><br><br />
2) Resuspend the cell suspension (for even distribution) in which you want to do the cell counting.<br> <br><br />
3) Take 10 ul from the cell suspension (using sterile tip), <br><br><br />
inject into the chamber(marked with arrow) by touching the border of the two glasses with the pipet tip.<br><br><br />
4) Place the chamber under the phase-contrast microscope and try to find the lines.<br><br><br />
<br />
- count inside 3 big squares (an example is on the next picture): <br><br><br />
<br />
<html><br />
<img src="https://static.igem.org/mediawiki/2010/c/c3/Burker2.jpg" alirgn="left"><br />
</html><br><br><br />
<br />
<br />
then we take the average of the 3 big squares, and it will give us the number of the cells in 0,1 ul<br><Br><br />
we take it 10 in the factor of 4 times and it will give us the cell number in 1 ml cell suspension.<br><br><br />
<br />
NOTE: Don't forget that the cell suspenson in the chamber is now not sterile,<br><br />
don't put it back into the cell solution. Clean the chamber with alcohol and water.<br><br />
<br />
<br />
1. To reach the appropriate confluency the day after plating, we put 50.000 cells into each well.<br><br />
For an 8 well chamber, if we calculate with 12 wells (because the volume loss), we put into a 15 ml centrifuge tube:<br><br />
- 50.000 x 12 = 600.000 cells [ in milliliter: counted cell number in 1 ml / 600.000 cells ]<br><br />
- we fill the cell suspension up to 8 x 300 ul = 2, 5 ml with 10% FBS DMEM<br><br />
(total volume of the wells are 300 ul)<Br><br><br />
2. With a 1 ml pipette put 300 ul from this suspension into each well. Sometimes invert the cell suspension containing tube <br>(the cells decent). After you finished the plate, swirl it circularly.<br><Br><br />
3. Incubate the cells for 1 day at 37°C, 5% CO2.<br><br><br />
<br />
<br />
<br />
<h5>2. FuGENE 6 transfection:</h5><Br><br />
The goal is to introduce foreign plasmid DNA into the plated COS1 cells 24 hours after the plating.<Br><Br><br />
<br />
Materials required:<Br><br />
- Plated cells in a LAB-TEK 8 well glass chamber slide ( with 50-80% confluency)<br><br />
- FBS/antibiotics/other additives Free DMEM medium<br><br />
- Sterile FuGENE 6 transfection reagent in a tightly capped glass vial<br><br />
- Plasmids on ice, in known concentrations: Beta-Gal (normalizer plasmid), Luciferase (tracer), Nuclear receptor, VDR- as <br>negative control, the plasmids are solved in sterile TE-buffer<br><br />
- 1,5 ml sterile Eppendorf tube, pipettes and tips<Br><br />
- Sterile laminar air flow box, 37°C incubator, 37°C waterbath<br><br />
- 70% ethanol squirt bottle, kimwipes<br><Br><br />
<br />
<br />
<h5>Steps:</h5><Br><br />
1. Prepare the sterile box: Open the sterile laminar box (Hood), turn on the ventillator and wait for 15 minutes to reach the optimal level of sterility (0,45 um filter). PUT ON YOUR GLOVES, spray down the base of the box with 70% ethanol, and wipe down with kimwipes.<br><br><br />
<br />
2. Spray down your hands, the DMEM medium, the glass Reagent vial and the pipettes and tips with 70% ethanol. Put these and the plasmids and the eppendorf tube into the sterile box.<br><Br><br />
<br />
3. Transfection mix reconstitution:<br><br />
<br />
&nbsp;&nbsp;3.1 Invert the room temp. FuGEGE 6 Transfection Reagent glass vial 2-3 times to distribute the components.<br><br />
&nbsp;&nbsp;3.2 Dilute the FuGENE reagent with Serume/antibiotics/other additives free DMEM medium – the order and manner of &nbsp;&nbsp;addition is critical:<Br><br />
&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Label a 1,5 ml eppendorf tube.<Br><br />
&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Pipet 75 ul FBS/antibiotics/other additives Free DMEM into the eppendorf tube.<Br><br />
&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Pipet 6 ul FuGENE 6 Transfection Reagent directly into the medium, without allowing contact between the plastic &nbsp;&nbsp;&nbsp;wall and the undiluted reagent.<br><br />
&nbsp;&nbsp;3.3 Vortex the mix for 1 second.<br><br />
&nbsp;&nbsp;3.4 Incubate the mix for 5 minutes at room temperature.<br><br />
&nbsp;&nbsp;3.5 Add 500 ng from each of the three plasmids (receptor, Luciferase, beta – Gal.) into the diluted FuGENE 6 &nbsp;&nbsp;transfection reagent.<br><br />
&nbsp;&nbsp;3.6 Vortex the Transfection Reagent:Plasmid mixture for 1 second.<Br><br />
&nbsp;&nbsp;3.7 Incubate the mixture for 20 minutes at room temperature.<br><br><br />
<br />
4. Remove glass chamber slide with plated cells from the incubator, place in the sterile box without spraying down with ethanol.<br><Br><br />
<br />
5. Note: We don’t need to remove the culturing medium (10% FBS containing DMEM) from the cells, it does not have any effect on <Br>the transfection efficiency.<br><br />
Add 9 ul Transfection mix in a dropwise manner to each well. Swirl the chamber slide to ensure distribution over the entire surface.<Br><Br><br />
<br />
6. Put on the cap of the slide chamber. Return the cells to the 37°C incubator until the assay for gene expression is to be<br> performed.<br><br />
Note: it is not necessary to remove and replace the transfection mixture-containing medium with fresh medium until the assay, only if you used FBS Free medium during the whole experiment (to avoid the cell starvation).<Br><br><br />
<br />
7. Clean up after yourself, place the FuGENE reagent to +2 - +8 °C and be sure if the cap is tightly turned on the Reagent.<Br><br><br />
<br />
<br />
<h5>NOTES:</h5><br><br />
- store the reagent at +2 - +8, with the lid very tightly closed, in the original glass vial.<Br><br />
- Do not allow the reagent to contact plastic walls (pipet directly into serum free medium) to keep the maximal biological activity.<Br><br />
- Do not use siliconized pipet tips and tubes.<Br><br />
- To prepare transfection complexes for larger experiments or parallel experiments, proportionally increase the quantity <br>according to the total surface area of the cell culture vessel being used. (ul FuGENE Reagent: ug DNA = 4:1, the used vessel in <Br>this case has a 79,21 cm2 surface area)<br><br />
<br />
<br />
<html><br />
<object style="height: 190px; width: 290px" ><param name="movie" value="http://www.youtube.com/v/r8DlyOCRJRs" ><param name="allowFullScreen" value="true" ><param name="allowScriptAccess" value="always" ><embed src="http://www.youtube.com/v/r8DlyOCRJRs" type="application/x-shockwave-flash" allowfullscreen="true" allowScriptAccess="always" width="290" height="190"></object><br />
</html><br />
&nbsp;&nbsp;<br />
<html><br />
<object style="height: 190px; width: 290px" ><param name="movie" value="http://www.youtube.com/v/IgRxD-Tf5Eo" ><param name="allowFullScreen" value="true" ><param name="allowScriptAccess" value="always" ><embed src="http://www.youtube.com/v/IgRxD-Tf5Eo" type="application/x-shockwave-flash" allowfullscreen="true" allowScriptAccess="always" width="290" height="190" ></object><br />
</html><br />
<Br><br><br />
==Notes & troubleshooting==<br />
==References==<br />
1. Horbinski C, Stachowiak MK, Higgins D, Finnegan SG. Polyethyleneimine-mediated transfection of cultured postmitotic neurons from rat sympathetic ganglia and adult human retina. .[http://www.biomedcentral.com/1471-2202/2/2 BMC Neurosci. 2001;2:2].<br />
<br />
2. Pollard H, Remy JS, Loussouarn G, Demolombe S, Behr JP, Escande D: Polyethylenimine but not cationic lipids promotes transgene delivery to the nucleus in mammalian cells.[http://www.jbc.org/content/273/13/7507.long J Biol Chem 1998], 273:7507-7511<br />
<br />
==Links==<br />
<br />
[http://www.youtube.com/user/debrecenigem2010#p/u/13/r8DlyOCRJRs Video I]<br />
<br />
[http://www.youtube.com/user/debrecenigem2010#p/u/12/IgRxD-Tf5Eo Video II]</div>Liorhttp://2010.igem.org/File:Burker2.jpgFile:Burker2.jpg2010-10-27T09:46:08Z<p>Lior: </p>
<hr />
<div></div>Liorhttp://2010.igem.org/File:Burker1.jpgFile:Burker1.jpg2010-10-27T09:45:34Z<p>Lior: </p>
<hr />
<div></div>Liorhttp://2010.igem.org/Team:Debrecen-Hungary/indicontibiTeam:Debrecen-Hungary/indicontibi2010-10-27T08:12:39Z<p>Lior: </p>
<hr />
<div>{{Template:Debrecen-Hungary_secret}} <br />
<br />
<br />
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<a href="https://2010.igem.org/Team:Debrecen-Hungary"><img src="https://static.igem.org/mediawiki/2010/9/96/Backbuttonbck.jpg" align="right"></a><br />
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<br />
='''Contibution to project by team members'''=<br />
<br />
*Students (in alphabetical order) <br />
**Timea Beregi: Cloning work, wiki writing, team image, protocols writing <br />
**Erika Berényi: I prepared some psB1A3-TRECMV-polyA plasmid constructs( cloning, transfection, PCR), and prepared western blots after transfection. I uploaded description from apoptosis parts to the wiki and partsregistry. <br />
**Shun Chieh Liu - took part in the cloning task initially, demonstrated some in silico work, scriptwriter of the video projects, modified the side project page in the wiki. <br />
**Lior Malka - Wiki design and programming, Culture team, Wiki Video Project design, protocols uploading and design, in silico design/modeling of the parts registry<br />
**Daniel Markovits - Wiki design and graphics, Help for teams PCR, Cell Culture and Luciferase teams, Video Project Narrator<br />
**Katalin Nagy - Cloning work<br />
**Lilla Ozgyin - Cloning work, coordinator of transfections, actress in video protocols, protocol writing, uploading a part, Parts Registry work, some wiki work, making photos of labwork. <br />
**Katalin Sandor- Cloning, transfections, luciferase measurements, extraction of contaminated environmental samples, uploading to the wiki, making photos about labwork. <br />
*Student Team Coordinators (in alphabetical order) <br />
**Bence Daniel- Supervisor of the whole labwork, optimization of the luciferase measurements and the two hybrid system, 3D protein structures, wiki writing.<br />
**Endre Kristof - Cloning (e.g. new expression vector from standard Biobrick parts), transfections and coordination in the wet lab. Participating in project selection, fund raising, in silico design, video and media project. Uploading materials of project description, future possibilities, parts etc. to the wiki and informations about the composite and some basic parts to the partsregistry.<br />
**Yakir Guri - Project selection and planning, cell culture team, cloning and in silico design. The supervisor of the video tutorial team (planning, film making, editing, soundtracks). Coordinator of PR and fundraising, writing articles to different newspapers (media, images, interviewing the team). As member of the wiki team coordinating the protocols formulation, reviewing, editing and writing into the OpenWetWare (online sourse).<br />
**Ophir Keret - Intiated project together with Balint.L.Balint, Team recruitment, project selection, work planning and coordination, team tutorials, labwork supervision, in silico design/modeling of the parts, construction of the expression vector, PCR work, Chief wiki editor, contributed to wiki writings (abstract, introduction, minimals, on the media). <br />
<br />
*Team Instructors (in alphabetical order) <br />
**Balint L. Balint - Initiated the project, made the team recruitment, fund raising, some of the PR, laboratory setup, helped shaping the project, seting up of the team. I supervised the first lab experiments and planned some of the experiments later on, ordered parts and reagents, organized finances, organized administrative issues of the trip. Supervised wiki and partsregistry work. <br />
**Peter Brazda <br />
**Mate Demeny <br />
**Gabor Zahuczky- Supported the initiation of the project, fund raising, organization of finances and administration. Participated in the design of parts and wet lab education. <br />
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='''Contibution to project by team members'''=<br />
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*Students (in alphabetical order) <br />
**Timea Beregi: Cloning work, wiki writing, team image, protocols writing <br />
**Erika Berényi: I prepared some psB1A3-TRECMV-polyA plasmid constructs( cloning, transfection, PCR), and prepared western blots after transfection. I uploaded description from apoptosis parts to the wiki and partsregistry. <br />
**Shun Chieh Liu - took part in the cloning task initially, demonstrated some in silico work, scriptwriter of the video projects, modified the side project page in the wiki. <br />
**Lior Malka - Wiki design and programming, Culture team, Wiki Video Project design, Parts Registry moderator, protocols uploading and design<br />
**Daniel Markovits - Wiki design and graphics, Help for teams PCR, Cell Culture and Luciferase teams, Video Project Narrator<br />
**Katalin Nagy - Cloning work<br />
**Lilla Ozgyin - Cloning work, coordinator of transfections, actress in video protocols, protocol writing, uploading a part, Parts Registry work, some wiki work, making photos of labwork. <br />
**Katalin Sandor- Cloning, transfections, luciferase measurements, extraction of contaminated environmental samples, uploading to the wiki, making photos about labwork. <br />
*Student Team Coordinators (in alphabetical order) <br />
**Bence Daniel- Supervisor of the whole labwork, optimization of the luciferase measurements and the two hybrid system, 3D protein structures, wiki writing.<br />
**Endre Kristof - Cloning (e.g. new expression vector from standard Biobrick parts), transfections and coordination in the wet lab. Participating in project selection, fund raising, in silico design, video and media project. Uploading materials of project description, future possibilities, parts etc. to the wiki and informations about the composite and some basic parts to the partsregistry.<br />
**Yakir Guri - Project selection and planning, cell culture team, cloning and in silico design. The supervisor of the video tutorial team (planning, film making, editing, soundtracks). Coordinator of PR and fundraising, writing articles to different newspapers (media, images, interviewing the team). As member of the wiki team coordinating the protocols formulation, reviewing, editing and writing into the OpenWetWare (online sourse).<br />
**Ophir Keret - Intiated project together with Balint.L.Balint, Team recruitment, project selection, work planning and coordination, team tutorials, labwork supervision, in silico design/modeling of the parts, construction of the expression vector, PCR work, Chief wiki editor, contributed to wiki writings (abstract, introduction, minimals, on the media). <br />
<br />
*Team Instructors (in alphabetical order) <br />
**Balint L. Balint - Initiated the project, made the team recruitment, fund raising, some of the PR, laboratory setup, helped shaping the project, seting up of the team. I supervised the first lab experiments and planned some of the experiments later on, ordered parts and reagents, organized finances, organized administrative issues of the trip. Supervised wiki and partsregistry work. <br />
**Peter Brazda <br />
**Mate Demeny <br />
**Gabor Zahuczky- Supported the initiation of the project, fund raising, organization of finances and administration. Participated in the design of parts and wet lab education. <br />
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</html></div>Liorhttp://2010.igem.org/Team:Debrecen-Hungary/protocolsTeam:Debrecen-Hungary/protocols2010-10-26T22:41:18Z<p>Lior: /* Online References At Openwetware */</p>
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=Welcome To Our Protocols=<br />
<br />
<h1> "Science is nothing else but the art of proper reproducibility. " </h1> <br><br />
The iGEM experience is not merely a project or a conference, but it was the way that most of our students got acquainted with the world of biological research laboratory. Pipettes, solutions, gels, electrodes, dishes and other scary machinery quickly filled our lives. <br />
From day one we saw the vast importance of teaching our students to keep a proper laboratory journal. As time passed and our project grew., and with growth sprouted the idea of keeping an electronic laboratory journal with texts and video’s depicting the proper way of doing our niche of science. And so, we present to you our combined effort a text and video version<br />
<br />
=='''Contents'''==<br />
<br />
'''Notebook protocols utilized by the bacterial work subteam '''<br />
<br />
[[Team:Debrecen-Hungary/protocols#Making Lurea's Broth|Making Lurea's Broth]] - [[Team:Debrecen-Hungary/protocols#Transformation of competent cells|Transformation of competent cells]] - [[Team:Debrecen-Hungary/protocols#Mini Prep|Mini Prep]] - [[Team:Debrecen-Hungary/protocols#Midi Prep|Midi Prep]]<br />
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'''Notebook protocols utilized by the molecular tools subteam '''<br />
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[[Team:Debrecen-Hungary/protocols#Restriction enzyme digestion|Restriction enzyme digestion]] - [[Team:Debrecen-Hungary/protocols#Cutting gel for PCR product purification |PCR Purification]] - [[Team:Debrecen-Hungary/protocols#PCR|PCR]] - [[Team:Debrecen-Hungary/protocols#Gel electrophoresis|Gel electrophoresis]] <br />
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'''Notebook protocols utilized by the tissue culture subteam'''<br />
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[[Team:Debrecen-Hungary/protocols#Cell Passaging|Cell Passaging]] - [[Team:Debrecen-Hungary/protocols#Media PEI Preparation |Media PEI Preparation ]] - [[Team:Debrecen-Hungary/protocols#Transfection|Transfection ]] - [[Team:Debrecen-Hungary/protocols#Ligand Treatment |Ligand Treatment ]]<br />
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'''Notebook protocols utilized by the Luciferase subteam'''<br />
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[[Team:Debrecen-Hungary/protocols#Measuring Luciferase activity with the Victor plate reader |Measuring Luciferase activity with the Victor plate reader ]]<br />
<br />
== Notebook protocols utilized by the bacterial work subteam ==<br />
<br />
=== Making Lurea's Broth===<br />
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[https://2010.igem.org/Team:Debrecen-Hungary/protocols/makinglb Read more...]<br />
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[[Team:Debrecen-Hungary/protocols#Notebook|[TOP]]]<br />
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=== Transformation of competent cells ===<br />
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<object style="height: 170px; width: 270px" ><param name="movie" value="http://www.youtube.com/v/8fg_ldCMyW8" ><param name="allowFullScreen" value="true" ><param name="allowScriptAccess" value="always" ><embed src="http://www.youtube.com/v/8fg_ldCMyW8" type="application/x-shockwave-flash" allowfullscreen="true" allowScriptAccess="always" width="270" height="170" ></object><br />
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Transformation is the process of introducing foreign DNA (e.g plasmids, BAC) into a bacterium. Bacterial cells intTransformation of competent cellso which foreign DNA can be transformed are called competent. Some bacteria are naturally competent (e.g B. subtilis), whereas others such as E. coli are not naturally competent. Non-competent cells can be made competent and then transformed via one of two main approaches; chemical transformation and electroporation. It is important to note we have tested transformations of the distribution kit with this protocol.[https://2010.igem.org/Team:Debrecen-Hungary/protocols/Transformation_of_competent_cells Read more...]<br />
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[[Team:Debrecen-Hungary/protocols#Notebook|[TOP]]]<br />
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=== Mini Prep ===<br />
This protocol is designed for purification of up to 20 μg of high-copy plasmid DNA from 1–5 ml overnight cultures of E. coli in LB (Luria-Bertani) medium [https://2010.igem.org/Team:Debrecen-Hungary/protocols/miniprep Read more...]<br />
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===Midi Prep===<br />
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[https://2010.igem.org/Team:Debrecen-Hungary/protocols/midiprep Read more...]<br />
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[[Team:Debrecen-Hungary/protocols#Notebook|[TOP]]]<br />
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== Notebook protocols utilized by the molecular tools subteam ==<br />
<br />
=== Restriction enzyme digestion ===<br />
<br />
BioBrick standard biological parts are flanked by well characterized upstream and downstream sequences which are technically not considered part of the BioBrick part (aka prefix and suffix). These up/down stream segments contain restriction sites for specific restriction enzymes, which allows for the simple creation of larger BioBrick parts by chaining together smaller ones in any desired order.<br />
[https://2010.igem.org/Team:Debrecen-Hungary/protocols/Restriction_biobrick_parts Read more...]<br />
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[[Team:Debrecen-Hungary/protocols#Notebook|[TOP]]]<br />
<br />
=== Cutting gel for PCR product purification ===<br />
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<object style="height: 170px; width: 270px" ><param name="movie" value="http://www.youtube.com/v/U74NaSx3rDY" ><param name="allowFullScreen" value="true" ><param name="allowScriptAccess" value="always" ><embed src="http://www.youtube.com/v/U74NaSx3rDY" type="application/x-shockwave-flash" allowfullscreen="true" allowScriptAccess="always" width="270" height="170" ></object><br />
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[https://2010.igem.org/Team:Debrecen-Hungary/protocols/PCR_purification_from_Agarose_Gel Read more...]<br />
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[[Team:Debrecen-Hungary/protocols#Notebook|[TOP]]]<br />
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=== PCR ===<br />
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<object style="height: 170px; width: 290px" ><param name="movie" value="http://www.youtube.com/v/vIbKpzlOAXA" ><param name="allowFullScreen" value="true" ><param name="allowScriptAccess" value="always" ><embed src="http://www.youtube.com/v/vIbKpzlOAXA" type="application/x-shockwave-flash" allowfullscreen="true" allowScriptAccess="always" width="290" height="170" ></object><br />
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[https://2010.igem.org/Team:Debrecen-Hungary/protocols/PCR Read more...]<br />
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[[Team:Debrecen-Hungary/protocols#Notebook|[TOP]]]<br />
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=== Gel electrophoresis ===<br />
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<object style="height: 170px; width: 270px" ><param name="movie" value="http://www.youtube.com/v/Q-kmMidAo3A" ><param name="allowFullScreen" value="true" ><param name="allowScriptAccess" value="always" ><embed src="http://www.youtube.com/v/Q-kmMidAo3A" type="application/x-shockwave-flash" allowfullscreen="true" allowScriptAccess="always" width="270" height="170" ></object><br />
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[https://2010.igem.org/Team:Debrecen-Hungary/protocols/Gel_electrophoresis Read more...]<br />
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[[Team:Debrecen-Hungary/protocols#Notebook|[TOP]]]<br />
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== Notebook protocols utilized by the tissue culture subteam ==<br />
<br />
=== Cell Passaging ===<br />
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Cell passaging or splitting is a technique that enables an individual to keep cells alive and growing under cultured conditions for extended periods of time. Cells should be passed when they are 90%-100% confluent. You have to do the cell passage on every second to fourth day (i.e. on every Monday, Wednesday and Friday).<br />
After reaching the confluency, the cells do not get enough nutrients and do not have enough place where they can extend. The colour of the medium switches from reddish-pink to orange or yellow which shows acidic metabolic products.<br />
[https://2010.igem.org/Team:Debrecen-Hungary/protocols/Cell_Passaging Read more...]<br />
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[[Team:Debrecen-Hungary/protocols#Notebook|[TOP]]]<br />
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=== Media PEI Preparation ===<br />
Eukaryotic cells, derived from multicellular animal eukaryotes, can be maintained in culturing medias. Aside from temperature and gas mixture, the most commonly varied factor in eucaryotic culture systems is the growth medium. Recipes for growth media can vary in pH, glucose concentration, growth factors, and the presence of other nutrients. The type of the media used depends on the type of the cell line. [https://2010.igem.org/Team:Debrecen-Hungary/protocols/_Media_PEI_Preparation Read more...]<br />
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=== Transfection ===<br />
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<object style="height: 170px; width: 270px" ><param name="movie" value="http://www.youtube.com/v/r8DlyOCRJRs" ><param name="allowFullScreen" value="true" ><param name="allowScriptAccess" value="always" ><embed src="http://www.youtube.com/v/r8DlyOCRJRs" type="application/x-shockwave-flash" allowfullscreen="true" allowScriptAccess="always" width="270" height="170" ></object><br />
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The following protocol is used to succesfully introduce foreign plasmid DNA into pluripotent cells through a chemical way, PEI mediated transfection. PEI stands for Poliethilenimine, a cationic polymer which can bind nucleic acids and helps the DNA to get into the cells by receptor-mediated endocytosis.<br />
[https://2010.igem.org/Team:Debrecen-Hungary/protocols/_Transfection Read more...]<br />
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[[Team:Debrecen-Hungary/protocols#Notebook|[TOP]]]<br />
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=== Ligand Treatment ===<br />
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<object style="height: 170px; width: 270px" ><param name="movie" value="http://www.youtube.com/v/XnNfkADMjA0" ><param name="allowFullScreen" value="true" ><param name="allowScriptAccess" value="always" ><embed src="http://www.youtube.com/v/XnNfkADMjA0" type="application/x-shockwave-flash" allowfullscreen="true" allowScriptAccess="always" width="270" height="170" ></object><br />
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Ligand treatment is a procedure when we add the appropriate ligand to the nuclear receptor (NR, transcription factor). The NR got into the cells through a previous transfection step. After treatment the ligand-binded NRs will dimerize and bind to the DNA at specific Nuclear receptor Response Elements and this will promote the gene expression of the downstream gene. In further examinations we detect the expression level of the target gene by Luciferase assay. [https://2010.igem.org/Team:Debrecen-Hungary/protocols/Ligand_Treatment Read more...]<br />
<br />
[[Team:Debrecen-Hungary/protocols#Notebook|[TOP]]]<br />
<br />
== Notebook protocols utilized by the Luciferase subteam ==<br />
<br />
=== Measuring Luciferase activity with the Victor plate reader ===<br />
<br />
he Wallac 1420 VICTOR2 is a multilabel, multitask plate reader designed to support the future demands of industrial and academic laboratories for multiple assay technologies on a single platform. An extended version of the successful Wallac VICTOR multilabel reader, the VICTOR2 allows immediate access to more than 10 counting modes, covering all of the main nonradioactive counting technologies. [https://2010.igem.org/Team:Debrecen-Hungary/protocols/_Enzymatic_activity_with_Victor_machine Read more...]<br />
<br />
[[Team:Debrecen-Hungary/protocols#Notebook|[TOP]]]<br />
<br />
==Online References At Openwetware==<br />
<br />
[https://2010.igem.org/Team:Debrecen-Hungary/protocols/makinglb ''Main article - Making Lurea's Broth'']<br />
<br />
[http://www.openwetware.org/wiki/IGEM:University_of_Debrecen:Transformation_of_competent_cells ''Main article - Transformation of competent cells'']<br />
<br />
[http://openwetware.org/wiki/Editing_IGEM:University_of_Debrecen:_Mini_Prep ''Main article - Mini prep'']<br />
<br />
[http://openwetware.org/wiki/IGEM:University_of_Debrecen:Restriction_biobrick_parts#Notes ''Main article - Restriction enzyme digestion'']<br />
<br />
[http://www.openwetware.org/wiki/IGEM:University_of_Debrecen:PCR_purification_from_Agarose_Gel ''Main article - PCR product purification / Gel purification''] <br><br />
<br />
[http://www.openwetware.org/wiki/IGEM:University_of_Debrecen:_Polymerase_Chain_Reaction_%28PCR%29 ''Main article - PCR''] <br><br />
<br />
[http://openwetware.org/wiki/IGEM:University_of_Debrecen:Gel_electrophoresis ''Main article - Gel electrophoresis'']<br />
<br />
[http://www.openwetware.org/wiki/IGEM:University_of_Debrecen:Cell_Passaging ''Main article - Cell passaging'']<br />
<br />
[http://openwetware.org/wiki/IGEM:University_of_Debrecen:_Media_PEI_Preparation ''Main article - Media PEI preparation'']<br />
<br />
[http://www.openwetware.org/wiki/IGEM:University_of_Debrecen:_Transfection ''Main article - Transfection'']<br />
<br />
[http://openwetware.org/wiki/IGEM:University_of_Debrecen:Ligand_Treatment ''Main article - Ligand treatment''] <br />
<br />
[http://openwetware.org/wiki/IGEM:University_of_Debrecen:_Enzymatic_activity_with_Victor_machine ''Main article - Measuring Luciferase activity with the Victor plate reader'']<br />
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</html></div>Liorhttp://2010.igem.org/Team:Debrecen-Hungary/protocolsTeam:Debrecen-Hungary/protocols2010-10-26T22:09:55Z<p>Lior: /* PCR */</p>
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=Welcome To Our Protocols=<br />
<br />
<h1> "Science is nothing else but the art of proper reproducibility. " </h1> <br><br />
The iGEM experience is not merely a project or a conference, but it was the way that most of our students got acquainted with the world of biological research laboratory. Pipettes, solutions, gels, electrodes, dishes and other scary machinery quickly filled our lives. <br />
From day one we saw the vast importance of teaching our students to keep a proper laboratory journal. As time passed and our project grew., and with growth sprouted the idea of keeping an electronic laboratory journal with texts and video’s depicting the proper way of doing our niche of science. And so, we present to you our combined effort a text and video version<br />
<br />
=='''Contents'''==<br />
<br />
'''Notebook protocols utilized by the bacterial work subteam '''<br />
<br />
[[Team:Debrecen-Hungary/protocols#Making Lurea's Broth|Making Lurea's Broth]] - [[Team:Debrecen-Hungary/protocols#Transformation of competent cells|Transformation of competent cells]] - [[Team:Debrecen-Hungary/protocols#Mini Prep|Mini Prep]] - [[Team:Debrecen-Hungary/protocols#Midi Prep|Midi Prep]]<br />
<br />
'''Notebook protocols utilized by the molecular tools subteam '''<br />
<br />
[[Team:Debrecen-Hungary/protocols#Restriction enzyme digestion|Restriction enzyme digestion]] - [[Team:Debrecen-Hungary/protocols#Cutting gel for PCR product purification |PCR Purification]] - [[Team:Debrecen-Hungary/protocols#PCR|PCR]] - [[Team:Debrecen-Hungary/protocols#Gel electrophoresis|Gel electrophoresis]] <br />
<br />
'''Notebook protocols utilized by the tissue culture subteam'''<br />
<br />
[[Team:Debrecen-Hungary/protocols#Cell Passaging|Cell Passaging]] - [[Team:Debrecen-Hungary/protocols#Media PEI Preparation |Media PEI Preparation ]] - [[Team:Debrecen-Hungary/protocols#Transfection|Transfection ]] - [[Team:Debrecen-Hungary/protocols#Ligand Treatment |Ligand Treatment ]]<br />
<br />
'''Notebook protocols utilized by the Luciferase subteam'''<br />
<br />
[[Team:Debrecen-Hungary/protocols#Measuring Luciferase activity with the Victor plate reader |Measuring Luciferase activity with the Victor plate reader ]]<br />
<br />
== Notebook protocols utilized by the bacterial work subteam ==<br />
<br />
=== Making Lurea's Broth===<br />
<br />
[https://2010.igem.org/Team:Debrecen-Hungary/protocols/makinglb Read more...]<br />
<br />
[[Team:Debrecen-Hungary/protocols#Notebook|[TOP]]]<br />
<br />
=== Transformation of competent cells ===<br />
<html><br />
<object style="height: 170px; width: 270px" ><param name="movie" value="http://www.youtube.com/v/8fg_ldCMyW8" ><param name="allowFullScreen" value="true" ><param name="allowScriptAccess" value="always" ><embed src="http://www.youtube.com/v/8fg_ldCMyW8" type="application/x-shockwave-flash" allowfullscreen="true" allowScriptAccess="always" width="270" height="170" ></object><br />
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Transformation is the process of introducing foreign DNA (e.g plasmids, BAC) into a bacterium. Bacterial cells intTransformation of competent cellso which foreign DNA can be transformed are called competent. Some bacteria are naturally competent (e.g B. subtilis), whereas others such as E. coli are not naturally competent. Non-competent cells can be made competent and then transformed via one of two main approaches; chemical transformation and electroporation. It is important to note we have tested transformations of the distribution kit with this protocol.[https://2010.igem.org/Team:Debrecen-Hungary/protocols/Transformation_of_competent_cells Read more...]<br />
<br />
[[Team:Debrecen-Hungary/protocols#Notebook|[TOP]]]<br />
<br />
=== Mini Prep ===<br />
This protocol is designed for purification of up to 20 μg of high-copy plasmid DNA from 1–5 ml overnight cultures of E. coli in LB (Luria-Bertani) medium [https://2010.igem.org/Team:Debrecen-Hungary/protocols/miniprep Read more...]<br />
<br />
===Midi Prep===<br />
<html><br />
<object style="height: 170px; width: 270px" ><param name="movie" value="http://www.youtube.com/v/4JyHe5gxihU" ><param name="allowFullScreen" value="true" ><param name="allowScriptAccess" value="always" ><embed src="http://www.youtube.com/v/4JyHe5gxihU" type="application/x-shockwave-flash" allowfullscreen="true" allowScriptAccess="always" width="270" height="170" ></object><br />
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[https://2010.igem.org/Team:Debrecen-Hungary/protocols/midiprep Read more...]<br />
<br />
[[Team:Debrecen-Hungary/protocols#Notebook|[TOP]]]<br />
<br />
== Notebook protocols utilized by the molecular tools subteam ==<br />
<br />
=== Restriction enzyme digestion ===<br />
<br />
BioBrick standard biological parts are flanked by well characterized upstream and downstream sequences which are technically not considered part of the BioBrick part (aka prefix and suffix). These up/down stream segments contain restriction sites for specific restriction enzymes, which allows for the simple creation of larger BioBrick parts by chaining together smaller ones in any desired order.<br />
[https://2010.igem.org/Team:Debrecen-Hungary/protocols/Restriction_biobrick_parts Read more...]<br />
<br />
[[Team:Debrecen-Hungary/protocols#Notebook|[TOP]]]<br />
<br />
=== Cutting gel for PCR product purification ===<br />
<html><br />
<object style="height: 170px; width: 270px" ><param name="movie" value="http://www.youtube.com/v/U74NaSx3rDY" ><param name="allowFullScreen" value="true" ><param name="allowScriptAccess" value="always" ><embed src="http://www.youtube.com/v/U74NaSx3rDY" type="application/x-shockwave-flash" allowfullscreen="true" allowScriptAccess="always" width="270" height="170" ></object><br />
</html><br />
<br />
[https://2010.igem.org/Team:Debrecen-Hungary/protocols/PCR_purification_from_Agarose_Gel Read more...]<br />
<br />
[[Team:Debrecen-Hungary/protocols#Notebook|[TOP]]]<br />
<br />
<br />
=== PCR ===<br />
<html><br />
<object style="height: 170px; width: 290px" ><param name="movie" value="http://www.youtube.com/v/vIbKpzlOAXA" ><param name="allowFullScreen" value="true" ><param name="allowScriptAccess" value="always" ><embed src="http://www.youtube.com/v/vIbKpzlOAXA" type="application/x-shockwave-flash" allowfullscreen="true" allowScriptAccess="always" width="290" height="170" ></object><br />
</html><br />
<br />
[https://2010.igem.org/Team:Debrecen-Hungary/protocols/PCR Read more...]<br />
<br />
[[Team:Debrecen-Hungary/protocols#Notebook|[TOP]]]<br />
<br />
=== Gel electrophoresis ===<br />
<html><br />
<object style="height: 170px; width: 270px" ><param name="movie" value="http://www.youtube.com/v/Q-kmMidAo3A" ><param name="allowFullScreen" value="true" ><param name="allowScriptAccess" value="always" ><embed src="http://www.youtube.com/v/Q-kmMidAo3A" type="application/x-shockwave-flash" allowfullscreen="true" allowScriptAccess="always" width="270" height="170" ></object><br />
</html><br />
<br />
[https://2010.igem.org/Team:Debrecen-Hungary/protocols/Gel_electrophoresis Read more...]<br />
<br />
[[Team:Debrecen-Hungary/protocols#Notebook|[TOP]]]<br />
<br />
== Notebook protocols utilized by the tissue culture subteam ==<br />
<br />
=== Cell Passaging ===<br />
<html><br />
<object style="height: 170px; width: 270px" ><param name="movie" value="http://www.youtube.com/v/7BniRI8C1PM" ><param name="allowFullScreen" value="true" ><param name="allowScriptAccess" value="always" ><embed src="http://www.youtube.com/v/7BniRI8C1PM" type="application/x-shockwave-flash" allowfullscreen="true" allowScriptAccess="always" width="270" height="170" ></object><br />
</html><br><br />
Cell passaging or splitting is a technique that enables an individual to keep cells alive and growing under cultured conditions for extended periods of time. Cells should be passed when they are 90%-100% confluent. You have to do the cell passage on every second to fourth day (i.e. on every Monday, Wednesday and Friday).<br />
After reaching the confluency, the cells do not get enough nutrients and do not have enough place where they can extend. The colour of the medium switches from reddish-pink to orange or yellow which shows acidic metabolic products.<br />
[https://2010.igem.org/Team:Debrecen-Hungary/protocols/Cell_Passaging Read more...]<br />
<br />
[[Team:Debrecen-Hungary/protocols#Notebook|[TOP]]]<br />
<br />
=== Media PEI Preparation ===<br />
Eukaryotic cells, derived from multicellular animal eukaryotes, can be maintained in culturing medias. Aside from temperature and gas mixture, the most commonly varied factor in eucaryotic culture systems is the growth medium. Recipes for growth media can vary in pH, glucose concentration, growth factors, and the presence of other nutrients. The type of the media used depends on the type of the cell line. [https://2010.igem.org/Team:Debrecen-Hungary/protocols/_Media_PEI_Preparation Read more...]<br />
<br />
=== Transfection ===<br />
<html><br />
<object style="height: 170px; width: 270px" ><param name="movie" value="http://www.youtube.com/v/r8DlyOCRJRs" ><param name="allowFullScreen" value="true" ><param name="allowScriptAccess" value="always" ><embed src="http://www.youtube.com/v/r8DlyOCRJRs" type="application/x-shockwave-flash" allowfullscreen="true" allowScriptAccess="always" width="270" height="170" ></object><br />
</html><br />
<br />
The following protocol is used to succesfully introduce foreign plasmid DNA into pluripotent cells through a chemical way, PEI mediated transfection. PEI stands for Poliethilenimine, a cationic polymer which can bind nucleic acids and helps the DNA to get into the cells by receptor-mediated endocytosis.<br />
[https://2010.igem.org/Team:Debrecen-Hungary/protocols/_Transfection Read more...]<br />
<br />
[[Team:Debrecen-Hungary/protocols#Notebook|[TOP]]]<br />
<br />
=== Ligand Treatment ===<br />
<html><br />
<object style="height: 170px; width: 270px" ><param name="movie" value="http://www.youtube.com/v/XnNfkADMjA0" ><param name="allowFullScreen" value="true" ><param name="allowScriptAccess" value="always" ><embed src="http://www.youtube.com/v/XnNfkADMjA0" type="application/x-shockwave-flash" allowfullscreen="true" allowScriptAccess="always" width="270" height="170" ></object><br />
</html><br />
Ligand treatment is a procedure when we add the appropriate ligand to the nuclear receptor (NR, transcription factor). The NR got into the cells through a previous transfection step. After treatment the ligand-binded NRs will dimerize and bind to the DNA at specific Nuclear receptor Response Elements and this will promote the gene expression of the downstream gene. In further examinations we detect the expression level of the target gene by Luciferase assay. [https://2010.igem.org/Team:Debrecen-Hungary/protocols/Ligand_Treatment Read more...]<br />
<br />
[[Team:Debrecen-Hungary/protocols#Notebook|[TOP]]]<br />
<br />
== Notebook protocols utilized by the Luciferase subteam ==<br />
<br />
=== Measuring Luciferase activity with the Victor plate reader ===<br />
<br />
he Wallac 1420 VICTOR2 is a multilabel, multitask plate reader designed to support the future demands of industrial and academic laboratories for multiple assay technologies on a single platform. An extended version of the successful Wallac VICTOR multilabel reader, the VICTOR2 allows immediate access to more than 10 counting modes, covering all of the main nonradioactive counting technologies. [https://2010.igem.org/Team:Debrecen-Hungary/protocols/_Enzymatic_activity_with_Victor_machine Read more...]<br />
<br />
[[Team:Debrecen-Hungary/protocols#Notebook|[TOP]]]<br />
<br />
==Online References At Openwetware==<br />
<br />
[https://2010.igem.org/Team:Debrecen-Hungary/protocols/makinglb ''Main article - Making Lurea's Broth'']<br />
<br />
[http://www.openwetware.org/wiki/IGEM:University_of_Debrecen:Transformation_of_competent_cells ''Main article - Transformation of competent cells'']<br />
<br />
[http://openwetware.org/wiki/Editing_IGEM:University_of_Debrecen:_Mini_Prep ''Main article - Mini prep'']<br />
<br />
[http://openwetware.org/wiki/IGEM:University_of_Debrecen:Restriction_biobrick_parts#Notes ''Main article - Restriction enzyme digestion'']<br />
<br />
[http://www.openwetware.org/wiki/IGEM:University_of_Debrecen:PCR_purification_from_Agarose_Gel ''Main article - PCR product purification / Gel purification''] <br><br />
<br />
[http://openwetware.org/wiki/IGEM:University_of_Debrecen:Gel_electrophoresis ''Main article - Gel electrophoresis'']<br />
<br />
[http://www.openwetware.org/wiki/IGEM:University_of_Debrecen:Cell_Passaging ''Main article - Cell passaging'']<br />
<br />
[http://openwetware.org/wiki/IGEM:University_of_Debrecen:_Media_PEI_Preparation ''Main article - Media PEI preparation'']<br />
<br />
[http://www.openwetware.org/wiki/IGEM:University_of_Debrecen:_Transfection ''Main article - Transfection'']<br />
<br />
[http://openwetware.org/wiki/IGEM:University_of_Debrecen:Ligand_Treatment ''Main article - Ligand treatment''] <br />
<br />
[http://openwetware.org/wiki/IGEM:University_of_Debrecen:_Enzymatic_activity_with_Victor_machine ''Main article - Measuring Luciferase activity with the Victor plate reader'']<br />
</div><br />
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<br />
<html><br />
<center><br />
<a href="https://2010.igem.org/Team:Debrecen-Hungary"><img src="https://static.igem.org/mediawiki/2010/9/96/Backbuttonbck.jpg"></a><br />
</center><br />
</html></div>Liorhttp://2010.igem.org/Team:Debrecen-Hungary/protocolsTeam:Debrecen-Hungary/protocols2010-10-26T22:09:20Z<p>Lior: /* PCR */</p>
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<img src="https://static.igem.org/mediawiki/2010/3/3a/Protocols.jpg" width="400" height="110"><br />
</td><br />
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<td align="right" bgcolor="#EDE8E2" border="0"><br />
<a href="https://2010.igem.org/Team:Debrecen-Hungary"><img src="https://static.igem.org/mediawiki/2010/9/96/Backbuttonbck.jpg" align="right"></a><br />
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<body id="Notebook"></body></html><br />
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<br />
<br />
=Welcome To Our Protocols=<br />
<br />
<h1> "Science is nothing else but the art of proper reproducibility. " </h1> <br><br />
The iGEM experience is not merely a project or a conference, but it was the way that most of our students got acquainted with the world of biological research laboratory. Pipettes, solutions, gels, electrodes, dishes and other scary machinery quickly filled our lives. <br />
From day one we saw the vast importance of teaching our students to keep a proper laboratory journal. As time passed and our project grew., and with growth sprouted the idea of keeping an electronic laboratory journal with texts and video’s depicting the proper way of doing our niche of science. And so, we present to you our combined effort a text and video version<br />
<br />
=='''Contents'''==<br />
<br />
'''Notebook protocols utilized by the bacterial work subteam '''<br />
<br />
[[Team:Debrecen-Hungary/protocols#Making Lurea's Broth|Making Lurea's Broth]] - [[Team:Debrecen-Hungary/protocols#Transformation of competent cells|Transformation of competent cells]] - [[Team:Debrecen-Hungary/protocols#Mini Prep|Mini Prep]] - [[Team:Debrecen-Hungary/protocols#Midi Prep|Midi Prep]]<br />
<br />
'''Notebook protocols utilized by the molecular tools subteam '''<br />
<br />
[[Team:Debrecen-Hungary/protocols#Restriction enzyme digestion|Restriction enzyme digestion]] - [[Team:Debrecen-Hungary/protocols#Cutting gel for PCR product purification |PCR Purification]] - [[Team:Debrecen-Hungary/protocols#PCR|PCR]] - [[Team:Debrecen-Hungary/protocols#Gel electrophoresis|Gel electrophoresis]] <br />
<br />
'''Notebook protocols utilized by the tissue culture subteam'''<br />
<br />
[[Team:Debrecen-Hungary/protocols#Cell Passaging|Cell Passaging]] - [[Team:Debrecen-Hungary/protocols#Media PEI Preparation |Media PEI Preparation ]] - [[Team:Debrecen-Hungary/protocols#Transfection|Transfection ]] - [[Team:Debrecen-Hungary/protocols#Ligand Treatment |Ligand Treatment ]]<br />
<br />
'''Notebook protocols utilized by the Luciferase subteam'''<br />
<br />
[[Team:Debrecen-Hungary/protocols#Measuring Luciferase activity with the Victor plate reader |Measuring Luciferase activity with the Victor plate reader ]]<br />
<br />
== Notebook protocols utilized by the bacterial work subteam ==<br />
<br />
=== Making Lurea's Broth===<br />
<br />
[https://2010.igem.org/Team:Debrecen-Hungary/protocols/makinglb Read more...]<br />
<br />
[[Team:Debrecen-Hungary/protocols#Notebook|[TOP]]]<br />
<br />
=== Transformation of competent cells ===<br />
<html><br />
<object style="height: 170px; width: 270px" ><param name="movie" value="http://www.youtube.com/v/8fg_ldCMyW8" ><param name="allowFullScreen" value="true" ><param name="allowScriptAccess" value="always" ><embed src="http://www.youtube.com/v/8fg_ldCMyW8" type="application/x-shockwave-flash" allowfullscreen="true" allowScriptAccess="always" width="270" height="170" ></object><br />
</html><br><br />
Transformation is the process of introducing foreign DNA (e.g plasmids, BAC) into a bacterium. Bacterial cells intTransformation of competent cellso which foreign DNA can be transformed are called competent. Some bacteria are naturally competent (e.g B. subtilis), whereas others such as E. coli are not naturally competent. Non-competent cells can be made competent and then transformed via one of two main approaches; chemical transformation and electroporation. It is important to note we have tested transformations of the distribution kit with this protocol.[https://2010.igem.org/Team:Debrecen-Hungary/protocols/Transformation_of_competent_cells Read more...]<br />
<br />
[[Team:Debrecen-Hungary/protocols#Notebook|[TOP]]]<br />
<br />
=== Mini Prep ===<br />
This protocol is designed for purification of up to 20 μg of high-copy plasmid DNA from 1–5 ml overnight cultures of E. coli in LB (Luria-Bertani) medium [https://2010.igem.org/Team:Debrecen-Hungary/protocols/miniprep Read more...]<br />
<br />
===Midi Prep===<br />
<html><br />
<object style="height: 170px; width: 270px" ><param name="movie" value="http://www.youtube.com/v/4JyHe5gxihU" ><param name="allowFullScreen" value="true" ><param name="allowScriptAccess" value="always" ><embed src="http://www.youtube.com/v/4JyHe5gxihU" type="application/x-shockwave-flash" allowfullscreen="true" allowScriptAccess="always" width="270" height="170" ></object><br />
</html><br />
<br />
[https://2010.igem.org/Team:Debrecen-Hungary/protocols/midiprep Read more...]<br />
<br />
[[Team:Debrecen-Hungary/protocols#Notebook|[TOP]]]<br />
<br />
== Notebook protocols utilized by the molecular tools subteam ==<br />
<br />
=== Restriction enzyme digestion ===<br />
<br />
BioBrick standard biological parts are flanked by well characterized upstream and downstream sequences which are technically not considered part of the BioBrick part (aka prefix and suffix). These up/down stream segments contain restriction sites for specific restriction enzymes, which allows for the simple creation of larger BioBrick parts by chaining together smaller ones in any desired order.<br />
[https://2010.igem.org/Team:Debrecen-Hungary/protocols/Restriction_biobrick_parts Read more...]<br />
<br />
[[Team:Debrecen-Hungary/protocols#Notebook|[TOP]]]<br />
<br />
=== Cutting gel for PCR product purification ===<br />
<html><br />
<object style="height: 170px; width: 270px" ><param name="movie" value="http://www.youtube.com/v/U74NaSx3rDY" ><param name="allowFullScreen" value="true" ><param name="allowScriptAccess" value="always" ><embed src="http://www.youtube.com/v/U74NaSx3rDY" type="application/x-shockwave-flash" allowfullscreen="true" allowScriptAccess="always" width="270" height="170" ></object><br />
</html><br />
<br />
[https://2010.igem.org/Team:Debrecen-Hungary/protocols/PCR_purification_from_Agarose_Gel Read more...]<br />
<br />
[[Team:Debrecen-Hungary/protocols#Notebook|[TOP]]]<br />
<br />
<br />
== PCR ==<br />
<html><br />
<object style="height: 170px; width: 290px" ><param name="movie" value="http://www.youtube.com/v/vIbKpzlOAXA" ><param name="allowFullScreen" value="true" ><param name="allowScriptAccess" value="always" ><embed src="http://www.youtube.com/v/vIbKpzlOAXA" type="application/x-shockwave-flash" allowfullscreen="true" allowScriptAccess="always" width="290" height="170" ></object><br />
</html><br />
<br />
[https://2010.igem.org/Team:Debrecen-Hungary/protocols/PCR Read more...]<br />
<br />
[[Team:Debrecen-Hungary/protocols#Notebook|[TOP]]]<br />
<br />
=== Gel electrophoresis ===<br />
<html><br />
<object style="height: 170px; width: 270px" ><param name="movie" value="http://www.youtube.com/v/Q-kmMidAo3A" ><param name="allowFullScreen" value="true" ><param name="allowScriptAccess" value="always" ><embed src="http://www.youtube.com/v/Q-kmMidAo3A" type="application/x-shockwave-flash" allowfullscreen="true" allowScriptAccess="always" width="270" height="170" ></object><br />
</html><br />
<br />
[https://2010.igem.org/Team:Debrecen-Hungary/protocols/Gel_electrophoresis Read more...]<br />
<br />
[[Team:Debrecen-Hungary/protocols#Notebook|[TOP]]]<br />
<br />
== Notebook protocols utilized by the tissue culture subteam ==<br />
<br />
=== Cell Passaging ===<br />
<html><br />
<object style="height: 170px; width: 270px" ><param name="movie" value="http://www.youtube.com/v/7BniRI8C1PM" ><param name="allowFullScreen" value="true" ><param name="allowScriptAccess" value="always" ><embed src="http://www.youtube.com/v/7BniRI8C1PM" type="application/x-shockwave-flash" allowfullscreen="true" allowScriptAccess="always" width="270" height="170" ></object><br />
</html><br><br />
Cell passaging or splitting is a technique that enables an individual to keep cells alive and growing under cultured conditions for extended periods of time. Cells should be passed when they are 90%-100% confluent. You have to do the cell passage on every second to fourth day (i.e. on every Monday, Wednesday and Friday).<br />
After reaching the confluency, the cells do not get enough nutrients and do not have enough place where they can extend. The colour of the medium switches from reddish-pink to orange or yellow which shows acidic metabolic products.<br />
[https://2010.igem.org/Team:Debrecen-Hungary/protocols/Cell_Passaging Read more...]<br />
<br />
[[Team:Debrecen-Hungary/protocols#Notebook|[TOP]]]<br />
<br />
=== Media PEI Preparation ===<br />
Eukaryotic cells, derived from multicellular animal eukaryotes, can be maintained in culturing medias. Aside from temperature and gas mixture, the most commonly varied factor in eucaryotic culture systems is the growth medium. Recipes for growth media can vary in pH, glucose concentration, growth factors, and the presence of other nutrients. The type of the media used depends on the type of the cell line. [https://2010.igem.org/Team:Debrecen-Hungary/protocols/_Media_PEI_Preparation Read more...]<br />
<br />
=== Transfection ===<br />
<html><br />
<object style="height: 170px; width: 270px" ><param name="movie" value="http://www.youtube.com/v/r8DlyOCRJRs" ><param name="allowFullScreen" value="true" ><param name="allowScriptAccess" value="always" ><embed src="http://www.youtube.com/v/r8DlyOCRJRs" type="application/x-shockwave-flash" allowfullscreen="true" allowScriptAccess="always" width="270" height="170" ></object><br />
</html><br />
<br />
The following protocol is used to succesfully introduce foreign plasmid DNA into pluripotent cells through a chemical way, PEI mediated transfection. PEI stands for Poliethilenimine, a cationic polymer which can bind nucleic acids and helps the DNA to get into the cells by receptor-mediated endocytosis.<br />
[https://2010.igem.org/Team:Debrecen-Hungary/protocols/_Transfection Read more...]<br />
<br />
[[Team:Debrecen-Hungary/protocols#Notebook|[TOP]]]<br />
<br />
=== Ligand Treatment ===<br />
<html><br />
<object style="height: 170px; width: 270px" ><param name="movie" value="http://www.youtube.com/v/XnNfkADMjA0" ><param name="allowFullScreen" value="true" ><param name="allowScriptAccess" value="always" ><embed src="http://www.youtube.com/v/XnNfkADMjA0" type="application/x-shockwave-flash" allowfullscreen="true" allowScriptAccess="always" width="270" height="170" ></object><br />
</html><br />
Ligand treatment is a procedure when we add the appropriate ligand to the nuclear receptor (NR, transcription factor). The NR got into the cells through a previous transfection step. After treatment the ligand-binded NRs will dimerize and bind to the DNA at specific Nuclear receptor Response Elements and this will promote the gene expression of the downstream gene. In further examinations we detect the expression level of the target gene by Luciferase assay. [https://2010.igem.org/Team:Debrecen-Hungary/protocols/Ligand_Treatment Read more...]<br />
<br />
[[Team:Debrecen-Hungary/protocols#Notebook|[TOP]]]<br />
<br />
== Notebook protocols utilized by the Luciferase subteam ==<br />
<br />
=== Measuring Luciferase activity with the Victor plate reader ===<br />
<br />
he Wallac 1420 VICTOR2 is a multilabel, multitask plate reader designed to support the future demands of industrial and academic laboratories for multiple assay technologies on a single platform. An extended version of the successful Wallac VICTOR multilabel reader, the VICTOR2 allows immediate access to more than 10 counting modes, covering all of the main nonradioactive counting technologies. [https://2010.igem.org/Team:Debrecen-Hungary/protocols/_Enzymatic_activity_with_Victor_machine Read more...]<br />
<br />
[[Team:Debrecen-Hungary/protocols#Notebook|[TOP]]]<br />
<br />
==Online References At Openwetware==<br />
<br />
[https://2010.igem.org/Team:Debrecen-Hungary/protocols/makinglb ''Main article - Making Lurea's Broth'']<br />
<br />
[http://www.openwetware.org/wiki/IGEM:University_of_Debrecen:Transformation_of_competent_cells ''Main article - Transformation of competent cells'']<br />
<br />
[http://openwetware.org/wiki/Editing_IGEM:University_of_Debrecen:_Mini_Prep ''Main article - Mini prep'']<br />
<br />
[http://openwetware.org/wiki/IGEM:University_of_Debrecen:Restriction_biobrick_parts#Notes ''Main article - Restriction enzyme digestion'']<br />
<br />
[http://www.openwetware.org/wiki/IGEM:University_of_Debrecen:PCR_purification_from_Agarose_Gel ''Main article - PCR product purification / Gel purification''] <br><br />
<br />
[http://openwetware.org/wiki/IGEM:University_of_Debrecen:Gel_electrophoresis ''Main article - Gel electrophoresis'']<br />
<br />
[http://www.openwetware.org/wiki/IGEM:University_of_Debrecen:Cell_Passaging ''Main article - Cell passaging'']<br />
<br />
[http://openwetware.org/wiki/IGEM:University_of_Debrecen:_Media_PEI_Preparation ''Main article - Media PEI preparation'']<br />
<br />
[http://www.openwetware.org/wiki/IGEM:University_of_Debrecen:_Transfection ''Main article - Transfection'']<br />
<br />
[http://openwetware.org/wiki/IGEM:University_of_Debrecen:Ligand_Treatment ''Main article - Ligand treatment''] <br />
<br />
[http://openwetware.org/wiki/IGEM:University_of_Debrecen:_Enzymatic_activity_with_Victor_machine ''Main article - Measuring Luciferase activity with the Victor plate reader'']<br />
</div><br />
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<br />
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<center><br />
<a href="https://2010.igem.org/Team:Debrecen-Hungary"><img src="https://static.igem.org/mediawiki/2010/9/96/Backbuttonbck.jpg"></a><br />
</center><br />
</html></div>Liorhttp://2010.igem.org/Team:Debrecen-Hungary/protocolsTeam:Debrecen-Hungary/protocols2010-10-26T22:09:01Z<p>Lior: /* PCR */</p>
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<a href="https://2010.igem.org/Team:Debrecen-Hungary"><img src="https://static.igem.org/mediawiki/2010/9/96/Backbuttonbck.jpg" align="right"></a><br />
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<br />
<body id="Notebook"></body></html><br />
<br />
<div align="justify"><br />
<br />
<br />
<br />
=Welcome To Our Protocols=<br />
<br />
<h1> "Science is nothing else but the art of proper reproducibility. " </h1> <br><br />
The iGEM experience is not merely a project or a conference, but it was the way that most of our students got acquainted with the world of biological research laboratory. Pipettes, solutions, gels, electrodes, dishes and other scary machinery quickly filled our lives. <br />
From day one we saw the vast importance of teaching our students to keep a proper laboratory journal. As time passed and our project grew., and with growth sprouted the idea of keeping an electronic laboratory journal with texts and video’s depicting the proper way of doing our niche of science. And so, we present to you our combined effort a text and video version<br />
<br />
=='''Contents'''==<br />
<br />
'''Notebook protocols utilized by the bacterial work subteam '''<br />
<br />
[[Team:Debrecen-Hungary/protocols#Making Lurea's Broth|Making Lurea's Broth]] - [[Team:Debrecen-Hungary/protocols#Transformation of competent cells|Transformation of competent cells]] - [[Team:Debrecen-Hungary/protocols#Mini Prep|Mini Prep]] - [[Team:Debrecen-Hungary/protocols#Midi Prep|Midi Prep]]<br />
<br />
'''Notebook protocols utilized by the molecular tools subteam '''<br />
<br />
[[Team:Debrecen-Hungary/protocols#Restriction enzyme digestion|Restriction enzyme digestion]] - [[Team:Debrecen-Hungary/protocols#Cutting gel for PCR product purification |PCR Purification]] - [[Team:Debrecen-Hungary/protocols#PCR|PCR]] - [[Team:Debrecen-Hungary/protocols#Gel electrophoresis|Gel electrophoresis]] <br />
<br />
'''Notebook protocols utilized by the tissue culture subteam'''<br />
<br />
[[Team:Debrecen-Hungary/protocols#Cell Passaging|Cell Passaging]] - [[Team:Debrecen-Hungary/protocols#Media PEI Preparation |Media PEI Preparation ]] - [[Team:Debrecen-Hungary/protocols#Transfection|Transfection ]] - [[Team:Debrecen-Hungary/protocols#Ligand Treatment |Ligand Treatment ]]<br />
<br />
'''Notebook protocols utilized by the Luciferase subteam'''<br />
<br />
[[Team:Debrecen-Hungary/protocols#Measuring Luciferase activity with the Victor plate reader |Measuring Luciferase activity with the Victor plate reader ]]<br />
<br />
== Notebook protocols utilized by the bacterial work subteam ==<br />
<br />
=== Making Lurea's Broth===<br />
<br />
[https://2010.igem.org/Team:Debrecen-Hungary/protocols/makinglb Read more...]<br />
<br />
[[Team:Debrecen-Hungary/protocols#Notebook|[TOP]]]<br />
<br />
=== Transformation of competent cells ===<br />
<html><br />
<object style="height: 170px; width: 270px" ><param name="movie" value="http://www.youtube.com/v/8fg_ldCMyW8" ><param name="allowFullScreen" value="true" ><param name="allowScriptAccess" value="always" ><embed src="http://www.youtube.com/v/8fg_ldCMyW8" type="application/x-shockwave-flash" allowfullscreen="true" allowScriptAccess="always" width="270" height="170" ></object><br />
</html><br><br />
Transformation is the process of introducing foreign DNA (e.g plasmids, BAC) into a bacterium. Bacterial cells intTransformation of competent cellso which foreign DNA can be transformed are called competent. Some bacteria are naturally competent (e.g B. subtilis), whereas others such as E. coli are not naturally competent. Non-competent cells can be made competent and then transformed via one of two main approaches; chemical transformation and electroporation. It is important to note we have tested transformations of the distribution kit with this protocol.[https://2010.igem.org/Team:Debrecen-Hungary/protocols/Transformation_of_competent_cells Read more...]<br />
<br />
[[Team:Debrecen-Hungary/protocols#Notebook|[TOP]]]<br />
<br />
=== Mini Prep ===<br />
This protocol is designed for purification of up to 20 μg of high-copy plasmid DNA from 1–5 ml overnight cultures of E. coli in LB (Luria-Bertani) medium [https://2010.igem.org/Team:Debrecen-Hungary/protocols/miniprep Read more...]<br />
<br />
===Midi Prep===<br />
<html><br />
<object style="height: 170px; width: 270px" ><param name="movie" value="http://www.youtube.com/v/4JyHe5gxihU" ><param name="allowFullScreen" value="true" ><param name="allowScriptAccess" value="always" ><embed src="http://www.youtube.com/v/4JyHe5gxihU" type="application/x-shockwave-flash" allowfullscreen="true" allowScriptAccess="always" width="270" height="170" ></object><br />
</html><br />
<br />
[https://2010.igem.org/Team:Debrecen-Hungary/protocols/midiprep Read more...]<br />
<br />
[[Team:Debrecen-Hungary/protocols#Notebook|[TOP]]]<br />
<br />
== Notebook protocols utilized by the molecular tools subteam ==<br />
<br />
=== Restriction enzyme digestion ===<br />
<br />
BioBrick standard biological parts are flanked by well characterized upstream and downstream sequences which are technically not considered part of the BioBrick part (aka prefix and suffix). These up/down stream segments contain restriction sites for specific restriction enzymes, which allows for the simple creation of larger BioBrick parts by chaining together smaller ones in any desired order.<br />
[https://2010.igem.org/Team:Debrecen-Hungary/protocols/Restriction_biobrick_parts Read more...]<br />
<br />
[[Team:Debrecen-Hungary/protocols#Notebook|[TOP]]]<br />
<br />
=== Cutting gel for PCR product purification ===<br />
<html><br />
<object style="height: 170px; width: 270px" ><param name="movie" value="http://www.youtube.com/v/U74NaSx3rDY" ><param name="allowFullScreen" value="true" ><param name="allowScriptAccess" value="always" ><embed src="http://www.youtube.com/v/U74NaSx3rDY" type="application/x-shockwave-flash" allowfullscreen="true" allowScriptAccess="always" width="270" height="170" ></object><br />
</html><br />
<br />
[https://2010.igem.org/Team:Debrecen-Hungary/protocols/PCR_purification_from_Agarose_Gel Read more...]<br />
<br />
[[Team:Debrecen-Hungary/protocols#Notebook|[TOP]]]<br />
<br />
<br />
== PCR ==<br />
<html><br />
<object style="height: 170px; width: 290px" ><param name="movie" value="http://www.youtube.com/v/vIbKpzlOAXA" ><param name="allowFullScreen" value="true" ><param name="allowScriptAccess" value="always" ><embed src="http://www.youtube.com/v/vIbKpzlOAXA" type="application/x-shockwave-flash" allowfullscreen="true" allowScriptAccess="always" width="290" height="170" ></object><br />
</html><br><br><br />
<br />
[https://2010.igem.org/Team:Debrecen-Hungary/protocols/PCR Read more...]<br />
<br />
[[Team:Debrecen-Hungary/protocols#Notebook|[TOP]]]<br />
<br />
=== Gel electrophoresis ===<br />
<html><br />
<object style="height: 170px; width: 270px" ><param name="movie" value="http://www.youtube.com/v/Q-kmMidAo3A" ><param name="allowFullScreen" value="true" ><param name="allowScriptAccess" value="always" ><embed src="http://www.youtube.com/v/Q-kmMidAo3A" type="application/x-shockwave-flash" allowfullscreen="true" allowScriptAccess="always" width="270" height="170" ></object><br />
</html><br />
<br />
[https://2010.igem.org/Team:Debrecen-Hungary/protocols/Gel_electrophoresis Read more...]<br />
<br />
[[Team:Debrecen-Hungary/protocols#Notebook|[TOP]]]<br />
<br />
== Notebook protocols utilized by the tissue culture subteam ==<br />
<br />
=== Cell Passaging ===<br />
<html><br />
<object style="height: 170px; width: 270px" ><param name="movie" value="http://www.youtube.com/v/7BniRI8C1PM" ><param name="allowFullScreen" value="true" ><param name="allowScriptAccess" value="always" ><embed src="http://www.youtube.com/v/7BniRI8C1PM" type="application/x-shockwave-flash" allowfullscreen="true" allowScriptAccess="always" width="270" height="170" ></object><br />
</html><br><br />
Cell passaging or splitting is a technique that enables an individual to keep cells alive and growing under cultured conditions for extended periods of time. Cells should be passed when they are 90%-100% confluent. You have to do the cell passage on every second to fourth day (i.e. on every Monday, Wednesday and Friday).<br />
After reaching the confluency, the cells do not get enough nutrients and do not have enough place where they can extend. The colour of the medium switches from reddish-pink to orange or yellow which shows acidic metabolic products.<br />
[https://2010.igem.org/Team:Debrecen-Hungary/protocols/Cell_Passaging Read more...]<br />
<br />
[[Team:Debrecen-Hungary/protocols#Notebook|[TOP]]]<br />
<br />
=== Media PEI Preparation ===<br />
Eukaryotic cells, derived from multicellular animal eukaryotes, can be maintained in culturing medias. Aside from temperature and gas mixture, the most commonly varied factor in eucaryotic culture systems is the growth medium. Recipes for growth media can vary in pH, glucose concentration, growth factors, and the presence of other nutrients. The type of the media used depends on the type of the cell line. [https://2010.igem.org/Team:Debrecen-Hungary/protocols/_Media_PEI_Preparation Read more...]<br />
<br />
=== Transfection ===<br />
<html><br />
<object style="height: 170px; width: 270px" ><param name="movie" value="http://www.youtube.com/v/r8DlyOCRJRs" ><param name="allowFullScreen" value="true" ><param name="allowScriptAccess" value="always" ><embed src="http://www.youtube.com/v/r8DlyOCRJRs" type="application/x-shockwave-flash" allowfullscreen="true" allowScriptAccess="always" width="270" height="170" ></object><br />
</html><br />
<br />
The following protocol is used to succesfully introduce foreign plasmid DNA into pluripotent cells through a chemical way, PEI mediated transfection. PEI stands for Poliethilenimine, a cationic polymer which can bind nucleic acids and helps the DNA to get into the cells by receptor-mediated endocytosis.<br />
[https://2010.igem.org/Team:Debrecen-Hungary/protocols/_Transfection Read more...]<br />
<br />
[[Team:Debrecen-Hungary/protocols#Notebook|[TOP]]]<br />
<br />
=== Ligand Treatment ===<br />
<html><br />
<object style="height: 170px; width: 270px" ><param name="movie" value="http://www.youtube.com/v/XnNfkADMjA0" ><param name="allowFullScreen" value="true" ><param name="allowScriptAccess" value="always" ><embed src="http://www.youtube.com/v/XnNfkADMjA0" type="application/x-shockwave-flash" allowfullscreen="true" allowScriptAccess="always" width="270" height="170" ></object><br />
</html><br />
Ligand treatment is a procedure when we add the appropriate ligand to the nuclear receptor (NR, transcription factor). The NR got into the cells through a previous transfection step. After treatment the ligand-binded NRs will dimerize and bind to the DNA at specific Nuclear receptor Response Elements and this will promote the gene expression of the downstream gene. In further examinations we detect the expression level of the target gene by Luciferase assay. [https://2010.igem.org/Team:Debrecen-Hungary/protocols/Ligand_Treatment Read more...]<br />
<br />
[[Team:Debrecen-Hungary/protocols#Notebook|[TOP]]]<br />
<br />
== Notebook protocols utilized by the Luciferase subteam ==<br />
<br />
=== Measuring Luciferase activity with the Victor plate reader ===<br />
<br />
he Wallac 1420 VICTOR2 is a multilabel, multitask plate reader designed to support the future demands of industrial and academic laboratories for multiple assay technologies on a single platform. An extended version of the successful Wallac VICTOR multilabel reader, the VICTOR2 allows immediate access to more than 10 counting modes, covering all of the main nonradioactive counting technologies. [https://2010.igem.org/Team:Debrecen-Hungary/protocols/_Enzymatic_activity_with_Victor_machine Read more...]<br />
<br />
[[Team:Debrecen-Hungary/protocols#Notebook|[TOP]]]<br />
<br />
==Online References At Openwetware==<br />
<br />
[https://2010.igem.org/Team:Debrecen-Hungary/protocols/makinglb ''Main article - Making Lurea's Broth'']<br />
<br />
[http://www.openwetware.org/wiki/IGEM:University_of_Debrecen:Transformation_of_competent_cells ''Main article - Transformation of competent cells'']<br />
<br />
[http://openwetware.org/wiki/Editing_IGEM:University_of_Debrecen:_Mini_Prep ''Main article - Mini prep'']<br />
<br />
[http://openwetware.org/wiki/IGEM:University_of_Debrecen:Restriction_biobrick_parts#Notes ''Main article - Restriction enzyme digestion'']<br />
<br />
[http://www.openwetware.org/wiki/IGEM:University_of_Debrecen:PCR_purification_from_Agarose_Gel ''Main article - PCR product purification / Gel purification''] <br><br />
<br />
[http://openwetware.org/wiki/IGEM:University_of_Debrecen:Gel_electrophoresis ''Main article - Gel electrophoresis'']<br />
<br />
[http://www.openwetware.org/wiki/IGEM:University_of_Debrecen:Cell_Passaging ''Main article - Cell passaging'']<br />
<br />
[http://openwetware.org/wiki/IGEM:University_of_Debrecen:_Media_PEI_Preparation ''Main article - Media PEI preparation'']<br />
<br />
[http://www.openwetware.org/wiki/IGEM:University_of_Debrecen:_Transfection ''Main article - Transfection'']<br />
<br />
[http://openwetware.org/wiki/IGEM:University_of_Debrecen:Ligand_Treatment ''Main article - Ligand treatment''] <br />
<br />
[http://openwetware.org/wiki/IGEM:University_of_Debrecen:_Enzymatic_activity_with_Victor_machine ''Main article - Measuring Luciferase activity with the Victor plate reader'']<br />
</div><br />
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<a href="https://2010.igem.org/Team:Debrecen-Hungary"><img src="https://static.igem.org/mediawiki/2010/9/96/Backbuttonbck.jpg"></a><br />
</center><br />
</html></div>Liorhttp://2010.igem.org/Team:Debrecen-Hungary/protocolsTeam:Debrecen-Hungary/protocols2010-10-26T22:01:18Z<p>Lior: /* Ligand Treatment */</p>
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<img src="https://static.igem.org/mediawiki/2010/3/3a/Protocols.jpg" width="400" height="110"><br />
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<br />
=Welcome To Our Protocols=<br />
<br />
<h1> "Science is nothing else but the art of proper reproducibility. " </h1> <br><br />
The iGEM experience is not merely a project or a conference, but it was the way that most of our students got acquainted with the world of biological research laboratory. Pipettes, solutions, gels, electrodes, dishes and other scary machinery quickly filled our lives. <br />
From day one we saw the vast importance of teaching our students to keep a proper laboratory journal. As time passed and our project grew., and with growth sprouted the idea of keeping an electronic laboratory journal with texts and video’s depicting the proper way of doing our niche of science. And so, we present to you our combined effort a text and video version<br />
<br />
=='''Contents'''==<br />
<br />
'''Notebook protocols utilized by the bacterial work subteam '''<br />
<br />
[[Team:Debrecen-Hungary/protocols#Making Lurea's Broth|Making Lurea's Broth]] - [[Team:Debrecen-Hungary/protocols#Transformation of competent cells|Transformation of competent cells]] - [[Team:Debrecen-Hungary/protocols#Mini Prep|Mini Prep]] - [[Team:Debrecen-Hungary/protocols#Midi Prep|Midi Prep]]<br />
<br />
'''Notebook protocols utilized by the molecular tools subteam '''<br />
<br />
[[Team:Debrecen-Hungary/protocols#Restriction enzyme digestion|Restriction enzyme digestion]] - [[Team:Debrecen-Hungary/protocols#Cutting gel for PCR product purification |PCR Purification]] - [[Team:Debrecen-Hungary/protocols#PCR|PCR]] - [[Team:Debrecen-Hungary/protocols#Gel electrophoresis|Gel electrophoresis]] <br />
<br />
'''Notebook protocols utilized by the tissue culture subteam'''<br />
<br />
[[Team:Debrecen-Hungary/protocols#Cell Passaging|Cell Passaging]] - [[Team:Debrecen-Hungary/protocols#Media PEI Preparation |Media PEI Preparation ]] - [[Team:Debrecen-Hungary/protocols#Transfection|Transfection ]] - [[Team:Debrecen-Hungary/protocols#Ligand Treatment |Ligand Treatment ]]<br />
<br />
'''Notebook protocols utilized by the Luciferase subteam'''<br />
<br />
[[Team:Debrecen-Hungary/protocols#Measuring Luciferase activity with the Victor plate reader |Measuring Luciferase activity with the Victor plate reader ]]<br />
<br />
== Notebook protocols utilized by the bacterial work subteam ==<br />
<br />
=== Making Lurea's Broth===<br />
<br />
[https://2010.igem.org/Team:Debrecen-Hungary/protocols/makinglb Read more...]<br />
<br />
[[Team:Debrecen-Hungary/protocols#Notebook|[TOP]]]<br />
<br />
=== Transformation of competent cells ===<br />
<html><br />
<object style="height: 170px; width: 270px" ><param name="movie" value="http://www.youtube.com/v/8fg_ldCMyW8" ><param name="allowFullScreen" value="true" ><param name="allowScriptAccess" value="always" ><embed src="http://www.youtube.com/v/8fg_ldCMyW8" type="application/x-shockwave-flash" allowfullscreen="true" allowScriptAccess="always" width="270" height="170" ></object><br />
</html><br><br />
Transformation is the process of introducing foreign DNA (e.g plasmids, BAC) into a bacterium. Bacterial cells intTransformation of competent cellso which foreign DNA can be transformed are called competent. Some bacteria are naturally competent (e.g B. subtilis), whereas others such as E. coli are not naturally competent. Non-competent cells can be made competent and then transformed via one of two main approaches; chemical transformation and electroporation. It is important to note we have tested transformations of the distribution kit with this protocol.[https://2010.igem.org/Team:Debrecen-Hungary/protocols/Transformation_of_competent_cells Read more...]<br />
<br />
[[Team:Debrecen-Hungary/protocols#Notebook|[TOP]]]<br />
<br />
=== Mini Prep ===<br />
This protocol is designed for purification of up to 20 μg of high-copy plasmid DNA from 1–5 ml overnight cultures of E. coli in LB (Luria-Bertani) medium [https://2010.igem.org/Team:Debrecen-Hungary/protocols/miniprep Read more...]<br />
<br />
===Midi Prep===<br />
<html><br />
<object style="height: 170px; width: 270px" ><param name="movie" value="http://www.youtube.com/v/4JyHe5gxihU" ><param name="allowFullScreen" value="true" ><param name="allowScriptAccess" value="always" ><embed src="http://www.youtube.com/v/4JyHe5gxihU" type="application/x-shockwave-flash" allowfullscreen="true" allowScriptAccess="always" width="270" height="170" ></object><br />
</html><br />
<br />
[https://2010.igem.org/Team:Debrecen-Hungary/protocols/midiprep Read more...]<br />
<br />
[[Team:Debrecen-Hungary/protocols#Notebook|[TOP]]]<br />
<br />
== Notebook protocols utilized by the molecular tools subteam ==<br />
<br />
=== Restriction enzyme digestion ===<br />
<br />
BioBrick standard biological parts are flanked by well characterized upstream and downstream sequences which are technically not considered part of the BioBrick part (aka prefix and suffix). These up/down stream segments contain restriction sites for specific restriction enzymes, which allows for the simple creation of larger BioBrick parts by chaining together smaller ones in any desired order.<br />
[https://2010.igem.org/Team:Debrecen-Hungary/protocols/Restriction_biobrick_parts Read more...]<br />
<br />
[[Team:Debrecen-Hungary/protocols#Notebook|[TOP]]]<br />
<br />
=== Cutting gel for PCR product purification ===<br />
<html><br />
<object style="height: 170px; width: 270px" ><param name="movie" value="http://www.youtube.com/v/U74NaSx3rDY" ><param name="allowFullScreen" value="true" ><param name="allowScriptAccess" value="always" ><embed src="http://www.youtube.com/v/U74NaSx3rDY" type="application/x-shockwave-flash" allowfullscreen="true" allowScriptAccess="always" width="270" height="170" ></object><br />
</html><br />
<br />
[https://2010.igem.org/Team:Debrecen-Hungary/protocols/PCR_purification_from_Agarose_Gel Read more...]<br />
<br />
[[Team:Debrecen-Hungary/protocols#Notebook|[TOP]]]<br />
<br />
<br />
== PCR ==<br />
<html><br />
<object style="height: 170px; width: 290px" ><param name="movie" value="http://www.youtube.com/v/vIbKpzlOAXA" ><param name="allowFullScreen" value="true" ><param name="allowScriptAccess" value="always" ><embed src="http://www.youtube.com/v/vIbKpzlOAXA" type="application/x-shockwave-flash" allowfullscreen="true" allowScriptAccess="always" width="290" height="170" ></object><br />
</html><br><br><br />
<br />
[https://2010.igem.org/Team:Debrecen-Hungary/protocols/PCR Read more...]<br />
<br />
[[Team:Debrecen-Hungary/protocols#Notebook|[TOP]]]<br />
<br />
=== Gel electrophoresis ===<br />
<html><br />
<object style="height: 170px; width: 270px" ><param name="movie" value="http://www.youtube.com/v/Q-kmMidAo3A" ><param name="allowFullScreen" value="true" ><param name="allowScriptAccess" value="always" ><embed src="http://www.youtube.com/v/Q-kmMidAo3A" type="application/x-shockwave-flash" allowfullscreen="true" allowScriptAccess="always" width="270" height="170" ></object><br />
</html><br />
<br />
<br />
[https://2010.igem.org/Team:Debrecen-Hungary/protocols/Gel_electrophoresis Read more...]<br />
<br />
[[Team:Debrecen-Hungary/protocols#Notebook|[TOP]]]<br />
<br />
== Notebook protocols utilized by the tissue culture subteam ==<br />
<br />
=== Cell Passaging ===<br />
<html><br />
<object style="height: 170px; width: 270px" ><param name="movie" value="http://www.youtube.com/v/7BniRI8C1PM" ><param name="allowFullScreen" value="true" ><param name="allowScriptAccess" value="always" ><embed src="http://www.youtube.com/v/7BniRI8C1PM" type="application/x-shockwave-flash" allowfullscreen="true" allowScriptAccess="always" width="270" height="170" ></object><br />
</html><br><br />
Cell passaging or splitting is a technique that enables an individual to keep cells alive and growing under cultured conditions for extended periods of time. Cells should be passed when they are 90%-100% confluent. You have to do the cell passage on every second to fourth day (i.e. on every Monday, Wednesday and Friday).<br />
After reaching the confluency, the cells do not get enough nutrients and do not have enough place where they can extend. The colour of the medium switches from reddish-pink to orange or yellow which shows acidic metabolic products.<br />
[https://2010.igem.org/Team:Debrecen-Hungary/protocols/Cell_Passaging Read more...]<br />
<br />
[[Team:Debrecen-Hungary/protocols#Notebook|[TOP]]]<br />
<br />
=== Media PEI Preparation ===<br />
Eukaryotic cells, derived from multicellular animal eukaryotes, can be maintained in culturing medias. Aside from temperature and gas mixture, the most commonly varied factor in eucaryotic culture systems is the growth medium. Recipes for growth media can vary in pH, glucose concentration, growth factors, and the presence of other nutrients. The type of the media used depends on the type of the cell line. [https://2010.igem.org/Team:Debrecen-Hungary/protocols/_Media_PEI_Preparation Read more...]<br />
<br />
=== Transfection ===<br />
<html><br />
<object style="height: 170px; width: 270px" ><param name="movie" value="http://www.youtube.com/v/r8DlyOCRJRs" ><param name="allowFullScreen" value="true" ><param name="allowScriptAccess" value="always" ><embed src="http://www.youtube.com/v/r8DlyOCRJRs" type="application/x-shockwave-flash" allowfullscreen="true" allowScriptAccess="always" width="270" height="170" ></object><br />
</html><br />
<br />
The following protocol is used to succesfully introduce foreign plasmid DNA into pluripotent cells through a chemical way, PEI mediated transfection. PEI stands for Poliethilenimine, a cationic polymer which can bind nucleic acids and helps the DNA to get into the cells by receptor-mediated endocytosis.<br />
[https://2010.igem.org/Team:Debrecen-Hungary/protocols/_Transfection Read more...]<br />
<br />
[[Team:Debrecen-Hungary/protocols#Notebook|[TOP]]]<br />
<br />
=== Ligand Treatment ===<br />
<html><br />
<object style="height: 170px; width: 270px" ><param name="movie" value="http://www.youtube.com/v/XnNfkADMjA0" ><param name="allowFullScreen" value="true" ><param name="allowScriptAccess" value="always" ><embed src="http://www.youtube.com/v/XnNfkADMjA0" type="application/x-shockwave-flash" allowfullscreen="true" allowScriptAccess="always" width="270" height="170" ></object><br />
</html><br />
Ligand treatment is a procedure when we add the appropriate ligand to the nuclear receptor (NR, transcription factor). The NR got into the cells through a previous transfection step. After treatment the ligand-binded NRs will dimerize and bind to the DNA at specific Nuclear receptor Response Elements and this will promote the gene expression of the downstream gene. In further examinations we detect the expression level of the target gene by Luciferase assay. [https://2010.igem.org/Team:Debrecen-Hungary/protocols/Ligand_Treatment Read more...]<br />
<br />
[[Team:Debrecen-Hungary/protocols#Notebook|[TOP]]]<br />
<br />
== Notebook protocols utilized by the Luciferase subteam ==<br />
<br />
=== Measuring Luciferase activity with the Victor plate reader ===<br />
<br />
he Wallac 1420 VICTOR2 is a multilabel, multitask plate reader designed to support the future demands of industrial and academic laboratories for multiple assay technologies on a single platform. An extended version of the successful Wallac VICTOR multilabel reader, the VICTOR2 allows immediate access to more than 10 counting modes, covering all of the main nonradioactive counting technologies. [https://2010.igem.org/Team:Debrecen-Hungary/protocols/_Enzymatic_activity_with_Victor_machine Read more...]<br />
<br />
[[Team:Debrecen-Hungary/protocols#Notebook|[TOP]]]<br />
<br />
==Online References At Openwetware==<br />
<br />
[https://2010.igem.org/Team:Debrecen-Hungary/protocols/makinglb ''Main article - Making Lurea's Broth'']<br />
<br />
[http://www.openwetware.org/wiki/IGEM:University_of_Debrecen:Transformation_of_competent_cells ''Main article - Transformation of competent cells'']<br />
<br />
[http://openwetware.org/wiki/Editing_IGEM:University_of_Debrecen:_Mini_Prep ''Main article - Mini prep'']<br />
<br />
[http://openwetware.org/wiki/IGEM:University_of_Debrecen:Restriction_biobrick_parts#Notes ''Main article - Restriction enzyme digestion'']<br />
<br />
[http://www.openwetware.org/wiki/IGEM:University_of_Debrecen:PCR_purification_from_Agarose_Gel ''Main article - PCR product purification / Gel purification''] <br><br />
<br />
[http://openwetware.org/wiki/IGEM:University_of_Debrecen:Gel_electrophoresis ''Main article - Gel electrophoresis'']<br />
<br />
[http://www.openwetware.org/wiki/IGEM:University_of_Debrecen:Cell_Passaging ''Main article - Cell passaging'']<br />
<br />
[http://openwetware.org/wiki/IGEM:University_of_Debrecen:_Media_PEI_Preparation ''Main article - Media PEI preparation'']<br />
<br />
[http://www.openwetware.org/wiki/IGEM:University_of_Debrecen:_Transfection ''Main article - Transfection'']<br />
<br />
[http://openwetware.org/wiki/IGEM:University_of_Debrecen:Ligand_Treatment ''Main article - Ligand treatment''] <br />
<br />
[http://openwetware.org/wiki/IGEM:University_of_Debrecen:_Enzymatic_activity_with_Victor_machine ''Main article - Measuring Luciferase activity with the Victor plate reader'']<br />
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</html></div>Liorhttp://2010.igem.org/Team:Debrecen-Hungary/protocolsTeam:Debrecen-Hungary/protocols2010-10-26T22:00:59Z<p>Lior: /* Ligand Treatment */</p>
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=Welcome To Our Protocols=<br />
<br />
<h1> "Science is nothing else but the art of proper reproducibility. " </h1> <br><br />
The iGEM experience is not merely a project or a conference, but it was the way that most of our students got acquainted with the world of biological research laboratory. Pipettes, solutions, gels, electrodes, dishes and other scary machinery quickly filled our lives. <br />
From day one we saw the vast importance of teaching our students to keep a proper laboratory journal. As time passed and our project grew., and with growth sprouted the idea of keeping an electronic laboratory journal with texts and video’s depicting the proper way of doing our niche of science. And so, we present to you our combined effort a text and video version<br />
<br />
=='''Contents'''==<br />
<br />
'''Notebook protocols utilized by the bacterial work subteam '''<br />
<br />
[[Team:Debrecen-Hungary/protocols#Making Lurea's Broth|Making Lurea's Broth]] - [[Team:Debrecen-Hungary/protocols#Transformation of competent cells|Transformation of competent cells]] - [[Team:Debrecen-Hungary/protocols#Mini Prep|Mini Prep]] - [[Team:Debrecen-Hungary/protocols#Midi Prep|Midi Prep]]<br />
<br />
'''Notebook protocols utilized by the molecular tools subteam '''<br />
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[[Team:Debrecen-Hungary/protocols#Restriction enzyme digestion|Restriction enzyme digestion]] - [[Team:Debrecen-Hungary/protocols#Cutting gel for PCR product purification |PCR Purification]] - [[Team:Debrecen-Hungary/protocols#PCR|PCR]] - [[Team:Debrecen-Hungary/protocols#Gel electrophoresis|Gel electrophoresis]] <br />
<br />
'''Notebook protocols utilized by the tissue culture subteam'''<br />
<br />
[[Team:Debrecen-Hungary/protocols#Cell Passaging|Cell Passaging]] - [[Team:Debrecen-Hungary/protocols#Media PEI Preparation |Media PEI Preparation ]] - [[Team:Debrecen-Hungary/protocols#Transfection|Transfection ]] - [[Team:Debrecen-Hungary/protocols#Ligand Treatment |Ligand Treatment ]]<br />
<br />
'''Notebook protocols utilized by the Luciferase subteam'''<br />
<br />
[[Team:Debrecen-Hungary/protocols#Measuring Luciferase activity with the Victor plate reader |Measuring Luciferase activity with the Victor plate reader ]]<br />
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== Notebook protocols utilized by the bacterial work subteam ==<br />
<br />
=== Making Lurea's Broth===<br />
<br />
[https://2010.igem.org/Team:Debrecen-Hungary/protocols/makinglb Read more...]<br />
<br />
[[Team:Debrecen-Hungary/protocols#Notebook|[TOP]]]<br />
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=== Transformation of competent cells ===<br />
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Transformation is the process of introducing foreign DNA (e.g plasmids, BAC) into a bacterium. Bacterial cells intTransformation of competent cellso which foreign DNA can be transformed are called competent. Some bacteria are naturally competent (e.g B. subtilis), whereas others such as E. coli are not naturally competent. Non-competent cells can be made competent and then transformed via one of two main approaches; chemical transformation and electroporation. It is important to note we have tested transformations of the distribution kit with this protocol.[https://2010.igem.org/Team:Debrecen-Hungary/protocols/Transformation_of_competent_cells Read more...]<br />
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[[Team:Debrecen-Hungary/protocols#Notebook|[TOP]]]<br />
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=== Mini Prep ===<br />
This protocol is designed for purification of up to 20 μg of high-copy plasmid DNA from 1–5 ml overnight cultures of E. coli in LB (Luria-Bertani) medium [https://2010.igem.org/Team:Debrecen-Hungary/protocols/miniprep Read more...]<br />
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===Midi Prep===<br />
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[https://2010.igem.org/Team:Debrecen-Hungary/protocols/midiprep Read more...]<br />
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[[Team:Debrecen-Hungary/protocols#Notebook|[TOP]]]<br />
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== Notebook protocols utilized by the molecular tools subteam ==<br />
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=== Restriction enzyme digestion ===<br />
<br />
BioBrick standard biological parts are flanked by well characterized upstream and downstream sequences which are technically not considered part of the BioBrick part (aka prefix and suffix). These up/down stream segments contain restriction sites for specific restriction enzymes, which allows for the simple creation of larger BioBrick parts by chaining together smaller ones in any desired order.<br />
[https://2010.igem.org/Team:Debrecen-Hungary/protocols/Restriction_biobrick_parts Read more...]<br />
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[[Team:Debrecen-Hungary/protocols#Notebook|[TOP]]]<br />
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=== Cutting gel for PCR product purification ===<br />
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<object style="height: 170px; width: 270px" ><param name="movie" value="http://www.youtube.com/v/U74NaSx3rDY" ><param name="allowFullScreen" value="true" ><param name="allowScriptAccess" value="always" ><embed src="http://www.youtube.com/v/U74NaSx3rDY" type="application/x-shockwave-flash" allowfullscreen="true" allowScriptAccess="always" width="270" height="170" ></object><br />
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[https://2010.igem.org/Team:Debrecen-Hungary/protocols/PCR_purification_from_Agarose_Gel Read more...]<br />
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[[Team:Debrecen-Hungary/protocols#Notebook|[TOP]]]<br />
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== PCR ==<br />
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<object style="height: 170px; width: 290px" ><param name="movie" value="http://www.youtube.com/v/vIbKpzlOAXA" ><param name="allowFullScreen" value="true" ><param name="allowScriptAccess" value="always" ><embed src="http://www.youtube.com/v/vIbKpzlOAXA" type="application/x-shockwave-flash" allowfullscreen="true" allowScriptAccess="always" width="290" height="170" ></object><br />
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[https://2010.igem.org/Team:Debrecen-Hungary/protocols/PCR Read more...]<br />
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[[Team:Debrecen-Hungary/protocols#Notebook|[TOP]]]<br />
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=== Gel electrophoresis ===<br />
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[https://2010.igem.org/Team:Debrecen-Hungary/protocols/Gel_electrophoresis Read more...]<br />
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[[Team:Debrecen-Hungary/protocols#Notebook|[TOP]]]<br />
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== Notebook protocols utilized by the tissue culture subteam ==<br />
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=== Cell Passaging ===<br />
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Cell passaging or splitting is a technique that enables an individual to keep cells alive and growing under cultured conditions for extended periods of time. Cells should be passed when they are 90%-100% confluent. You have to do the cell passage on every second to fourth day (i.e. on every Monday, Wednesday and Friday).<br />
After reaching the confluency, the cells do not get enough nutrients and do not have enough place where they can extend. The colour of the medium switches from reddish-pink to orange or yellow which shows acidic metabolic products.<br />
[https://2010.igem.org/Team:Debrecen-Hungary/protocols/Cell_Passaging Read more...]<br />
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[[Team:Debrecen-Hungary/protocols#Notebook|[TOP]]]<br />
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=== Media PEI Preparation ===<br />
Eukaryotic cells, derived from multicellular animal eukaryotes, can be maintained in culturing medias. Aside from temperature and gas mixture, the most commonly varied factor in eucaryotic culture systems is the growth medium. Recipes for growth media can vary in pH, glucose concentration, growth factors, and the presence of other nutrients. The type of the media used depends on the type of the cell line. [https://2010.igem.org/Team:Debrecen-Hungary/protocols/_Media_PEI_Preparation Read more...]<br />
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=== Transfection ===<br />
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The following protocol is used to succesfully introduce foreign plasmid DNA into pluripotent cells through a chemical way, PEI mediated transfection. PEI stands for Poliethilenimine, a cationic polymer which can bind nucleic acids and helps the DNA to get into the cells by receptor-mediated endocytosis.<br />
[https://2010.igem.org/Team:Debrecen-Hungary/protocols/_Transfection Read more...]<br />
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[[Team:Debrecen-Hungary/protocols#Notebook|[TOP]]]<br />
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=== Ligand Treatment ===<br />
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Ligand treatment is a procedure when we add the appropriate ligand to the nuclear receptor (NR, transcription factor). The NR got into the cells through a previous transfection step. After treatment the ligand-binded NRs will dimerize and bind to the DNA at specific Nuclear receptor Response Elements and this will promote the gene expression of the downstream gene. In further examinations we detect the expression level of the target gene by Luciferase assay. [https://2010.igem.org/Team:Debrecen-Hungary/protocols/Ligand_Treatment Read more...]<br />
<br />
[[Team:Debrecen-Hungary/protocols#Notebook|[TOP]]]<br />
<br />
== Notebook protocols utilized by the Luciferase subteam ==<br />
<br />
=== Measuring Luciferase activity with the Victor plate reader ===<br />
<br />
he Wallac 1420 VICTOR2 is a multilabel, multitask plate reader designed to support the future demands of industrial and academic laboratories for multiple assay technologies on a single platform. An extended version of the successful Wallac VICTOR multilabel reader, the VICTOR2 allows immediate access to more than 10 counting modes, covering all of the main nonradioactive counting technologies. [https://2010.igem.org/Team:Debrecen-Hungary/protocols/_Enzymatic_activity_with_Victor_machine Read more...]<br />
<br />
[[Team:Debrecen-Hungary/protocols#Notebook|[TOP]]]<br />
<br />
==Online References At Openwetware==<br />
<br />
[https://2010.igem.org/Team:Debrecen-Hungary/protocols/makinglb ''Main article - Making Lurea's Broth'']<br />
<br />
[http://www.openwetware.org/wiki/IGEM:University_of_Debrecen:Transformation_of_competent_cells ''Main article - Transformation of competent cells'']<br />
<br />
[http://openwetware.org/wiki/Editing_IGEM:University_of_Debrecen:_Mini_Prep ''Main article - Mini prep'']<br />
<br />
[http://openwetware.org/wiki/IGEM:University_of_Debrecen:Restriction_biobrick_parts#Notes ''Main article - Restriction enzyme digestion'']<br />
<br />
[http://www.openwetware.org/wiki/IGEM:University_of_Debrecen:PCR_purification_from_Agarose_Gel ''Main article - PCR product purification / Gel purification''] <br><br />
<br />
[http://openwetware.org/wiki/IGEM:University_of_Debrecen:Gel_electrophoresis ''Main article - Gel electrophoresis'']<br />
<br />
[http://www.openwetware.org/wiki/IGEM:University_of_Debrecen:Cell_Passaging ''Main article - Cell passaging'']<br />
<br />
[http://openwetware.org/wiki/IGEM:University_of_Debrecen:_Media_PEI_Preparation ''Main article - Media PEI preparation'']<br />
<br />
[http://www.openwetware.org/wiki/IGEM:University_of_Debrecen:_Transfection ''Main article - Transfection'']<br />
<br />
[http://openwetware.org/wiki/IGEM:University_of_Debrecen:Ligand_Treatment ''Main article - Ligand treatment''] <br />
<br />
[http://openwetware.org/wiki/IGEM:University_of_Debrecen:_Enzymatic_activity_with_Victor_machine ''Main article - Measuring Luciferase activity with the Victor plate reader'']<br />
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</html></div>Liorhttp://2010.igem.org/Team:Debrecen-Hungary/protocols/Ligand_TreatmentTeam:Debrecen-Hungary/protocols/Ligand Treatment2010-10-26T21:55:57Z<p>Lior: /* Other */</p>
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==Scientific Background==<br />
<br />
Ligand treatment is a procedure when we add the appropriate ligand to the nuclear receptor (NR, transcription factor). The NR got into the cells through a previous transfection step. After treatment the ligand-binded NRs will dimerize and bind to the DNA at specific Nuclear receptor Response Elements and this will promote the gene expression of the downstream gene. In further examinations we detect the expression level of the target gene by Luciferase assay.<br />
<br />
==Overview==<br />
<br />
The target cells in our case are COS1 cells. Performing the protocol from the beginning to the end takes no more than 1 hour, and the following steps are included:<br />
<br />
1. Preparation of the ligand solutions<br />
<br />
2. Removal of the transfection medium<br />
<br />
The goal is to get rid of the transfection medium from the COS1 cells 5-7 hours after transfection, because:<br />
<br />
- PEI can damage the cells <br />
<br />
- FBS Free medium used for transfection can cause starvation<br />
<br />
3. Cell refeeding with Ligand-containing medium<br />
<br />
Our aim is to refeed the cells with 10% FBS containing DMEM and also give appropriate amounts of Ligand in one step.<br />
The 10% FBS in the medium will provide the necessary proteins and other molecules for proliferation and for life<br />
functions. <br />
The ligand will activate the nuclear receptor and will promote the gene expression.<br />
<br />
==Preparation of the ligand solutions==<br />
<br />
==Materials==<br />
<br />
- Nuclear receptor-transfected cells in a 48-well plate<br />
<br />
- 10% FBS containing DMEM medium<br />
<br />
- Ligand solution in known concentration<br />
<br />
- 50 ml, 15 ml tubes<br />
<br />
- Pasteur pipettes, pipettor, serological pipettes, pipettes, pipet tips<br />
<br />
- Sterile laminar air flow box with flame and vacuum system, 37°C waterbath, 37°C incubator, 70% ethanol squirt bottles<br />
<br />
<br />
==Procedure==<br />
<br />
'''- in the case of 1-type receptor transfection (GAL4PPARg):'''<br />
<br />
''We use different concentrations of ligand, so as to make a dose-response curve in the subsequent measuring process:<br />
''<br />
<br />
''10 μM:'' 200 * (x/10) μL Ligand up to 200μL with 10% DMEM /per well/<br />
<br />
''2 μM:'' 200 * (x/2) μL Ligand up to 200μL with 10%FBS DMEM /per well/<br />
<br />
''0,4 μM:'' 200 * (x/0,4) μL Ligand up to 200μL with 10%FBS DMEM /per well/<br />
<br />
''0,08 μM:'' 200 * (x/0,08) μL Ligand up to 200μL with 10%FBS DMEM /per well/<br />
<br />
''0,016 μM:'' 200 * (x/0,016) μL Ligand up to 200μL with 10%FBS DMEM /per well/<br />
<br />
''0 μM(negative control):'' 1 μL DMSO-ethanol(1:1) up to 200μL with 10%FBS DMEM /per well/<br />
<br />
if<br />
<br />
x = original ligand concentartion[mM]<br />
<br />
we use 48 well plates<br />
<br />
DMSO-ethanol is the dissolvent of the ligand<br />
<br />
<br />
''-Prepare the Ligand dilutions:''<br />
<br />
- We use 6 wells for 1 concentration / plate<br />
<br />
- the total concentration of one well is 200 μL<br />
<br />
- For this reason, from 1 concentration we need 6x200=1200 μL<br />
<br />
''We are doing serial dilutions (5-fold-dilutions) in 15 ml tubes:''<br />
<br />
1st dilution (10μM): I calculate with 5000 ul, put 5000*(x/10) μL Ligand solution and fill it up to 5000 μL with 10% FBS containing DMEM. Mix it with pipetting.<br />
<br />
2nd dilution (2μM): 1 ml from the 1st dilution + 4 ml DMEM 10% <br />
<br />
3rd dilution (0,4μM): 1 ml from the 2nd dilution + 4 ml DMEM 10% <br />
<br />
4th dilution (0,08μM): 1 ml from the 3rd dilution + 4 ml DMEM 10%<br />
<br />
5th dilution (0,016μM): 1 ml from the 4th dilution + 4 ml DMEM 10%<br />
<br />
6th solution (0 μM): 6 μL-ethanol 1:1 solution + 4,994 ml DMEM 10%<br />
<br />
*you can use serological pipettes and pipettor, or micropipettes with sterile pipette tips.<br />
<br />
*these solutions will be enough for three 48-well plates, if you have less than 3 plates you can store the tubes at +4°C and use them next time.<br />
<br />
<br />
----<br />
<br />
<br />
'''- in the case of a 2-type receptor transfection:'''<br />
<br />
The receptor to be activated is Vitamin D Receptor (VDR) and the Ligand is Vitamin D. We use 10e-8 μM Vitamin D. We treat 24 wells only, the other 24 will be negative controls.<br />
<br />
-Prepare the Vitamin D solution in a 15 ml tube:<br />
<br />
Fill ''2000 / (original cc. of the ligand solution [mM]/10e-8)'' μL ligand solution up to 2000 μL with 10% FBS containing DMEM. Use micropipettes and sterile pipet tips.<br />
<br />
==Removal of the transfection medium==<br />
==Materials==<br />
<br />
- Nuclear receptor-transfected cells in a 48-well plate<br />
<br />
- Pasteur pipettes <br />
<br />
- Sterile box with flame and vacuum system<br />
<br />
==Procedure==<br />
Steps:<br />
<br />
1. Prepare the hood : put on your gloves, turn on the ventillation (it needs 15 minutes to filter the air in the box), clean the inner space of the hood with 70% ethanol. Spray hands with ethanol.<br />
<br />
2. Place the plate into the hood.Turn on the vacuum system, insert the Pasteur pipette into the vacuum tube. Fire-sterilize the Pasteur pipette.<br />
<br />
3. Aspire the used medium from all of the wells, pay attention so as not to aspire the cells (touch only the bottom-side of the wells).<br />
<br />
4. Put back the cap of the plate, we don’t want the cells to go dry.<br />
<br />
==Cell refeeding with Ligand-containing medium==<br />
==Materials==<br />
<br />
- Ligand solutions prepared in point 1.<br />
<br />
- 48 well plate without medium (point 2.)<br />
<br />
- 10% FBS containing medium<br />
<br />
- Repeating pipet and tips<br />
<br />
- 37°C incubator, 37°C waterbath, Sterile laminar air flow box, 70% ethanol squirt bottles<br />
<br />
==Procedure==<br />
<br />
Steps:<br />
<br />
1. Warm up the Ligand solutions for 1- receptor transfected cells and the Ligand solution + 10% FBS DMEM for 2-receptor transfected cells<br />
<br />
2. You use the same hood which you prapared: put everything what is needed into the hood after spraying down with 70% alcohol.<br />
<br />
3. Take down the cap of the plate. Vortex the Ligand solutions, after that put <br />
200-200 ul from these solutions into the adequate wells quickly (in other case the cells will go dry). Release the solutions onto the wall of the wells<br />
<br />
After 1-receptor transfection - by using a 200 μl or a 1 ml pipet with sterile tips<br />
<br />
10 mM: A1-A3 and E1-E3<br />
<br />
2 mM: A4-A6 and E4-E6<br />
<br />
0,4 mM: B1-B3 and F1-F3<br />
<br />
0,08 mM: B4-B6 and F4-F6<br />
<br />
0,016 mM: C1-C3 and G1-G3<br />
<br />
0 mM: C4-C6 and G4-G6<br />
<br />
After 2-receptor transfection – by using a repeating pipet<br />
<br />
* Ligand treated /well A1-D6/ : 200 ul 10e-8 mM Ligand- DMEM solution<br />
<br />
* Non-treated /well E1-H6/ : 200 ul "0 μM" solution<br />
<br />
<br />
4. Put the „treated” COS1 cells into the 37°C incubator for 2 days, after 2 days comes the Luciferase assay to make sure that the transfection worked and to measure the Nuclear Receptor’s response to the ligand.<br />
<br />
5. Clean the surface<br />
<br />
==Notes & troubleshooting==<br />
<br />
==References==<br />
1. Junn Yanagisawa, Yasuo Yanagi, Yoshikazu Masuhiro, Miyuki Suzawa, Michiko Watanabe, Kouji Kashiwagi, Takeshi Toriyabe, Masahiro Kawabata, Kohei Miyazono, Shigeaki Kato "Convergence of Transforming Growth Factor- and Vitamin D Signaling Pathways on SMAD Transcriptional Coactivators" [http://www.sciencemag.org/cgi/content/abstract/283/5406/1317 Science 26 February 1999]<br />
<br />
==Video==<br />
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</html></div>Liorhttp://2010.igem.org/Team:Debrecen-Hungary/protocolsTeam:Debrecen-Hungary/protocols2010-10-26T21:54:27Z<p>Lior: /* Transfection */</p>
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=Welcome To Our Protocols=<br />
<br />
<h1> "Science is nothing else but the art of proper reproducibility. " </h1> <br><br />
The iGEM experience is not merely a project or a conference, but it was the way that most of our students got acquainted with the world of biological research laboratory. Pipettes, solutions, gels, electrodes, dishes and other scary machinery quickly filled our lives. <br />
From day one we saw the vast importance of teaching our students to keep a proper laboratory journal. As time passed and our project grew., and with growth sprouted the idea of keeping an electronic laboratory journal with texts and video’s depicting the proper way of doing our niche of science. And so, we present to you our combined effort a text and video version<br />
<br />
=='''Contents'''==<br />
<br />
'''Notebook protocols utilized by the bacterial work subteam '''<br />
<br />
[[Team:Debrecen-Hungary/protocols#Making Lurea's Broth|Making Lurea's Broth]] - [[Team:Debrecen-Hungary/protocols#Transformation of competent cells|Transformation of competent cells]] - [[Team:Debrecen-Hungary/protocols#Mini Prep|Mini Prep]] - [[Team:Debrecen-Hungary/protocols#Midi Prep|Midi Prep]]<br />
<br />
'''Notebook protocols utilized by the molecular tools subteam '''<br />
<br />
[[Team:Debrecen-Hungary/protocols#Restriction enzyme digestion|Restriction enzyme digestion]] - [[Team:Debrecen-Hungary/protocols#Cutting gel for PCR product purification |PCR Purification]] - [[Team:Debrecen-Hungary/protocols#PCR|PCR]] - [[Team:Debrecen-Hungary/protocols#Gel electrophoresis|Gel electrophoresis]] <br />
<br />
'''Notebook protocols utilized by the tissue culture subteam'''<br />
<br />
[[Team:Debrecen-Hungary/protocols#Cell Passaging|Cell Passaging]] - [[Team:Debrecen-Hungary/protocols#Media PEI Preparation |Media PEI Preparation ]] - [[Team:Debrecen-Hungary/protocols#Transfection|Transfection ]] - [[Team:Debrecen-Hungary/protocols#Ligand Treatment |Ligand Treatment ]]<br />
<br />
'''Notebook protocols utilized by the Luciferase subteam'''<br />
<br />
[[Team:Debrecen-Hungary/protocols#Measuring Luciferase activity with the Victor plate reader |Measuring Luciferase activity with the Victor plate reader ]]<br />
<br />
== Notebook protocols utilized by the bacterial work subteam ==<br />
<br />
=== Making Lurea's Broth===<br />
<br />
[https://2010.igem.org/Team:Debrecen-Hungary/protocols/makinglb Read more...]<br />
<br />
[[Team:Debrecen-Hungary/protocols#Notebook|[TOP]]]<br />
<br />
=== Transformation of competent cells ===<br />
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Transformation is the process of introducing foreign DNA (e.g plasmids, BAC) into a bacterium. Bacterial cells intTransformation of competent cellso which foreign DNA can be transformed are called competent. Some bacteria are naturally competent (e.g B. subtilis), whereas others such as E. coli are not naturally competent. Non-competent cells can be made competent and then transformed via one of two main approaches; chemical transformation and electroporation. It is important to note we have tested transformations of the distribution kit with this protocol.[https://2010.igem.org/Team:Debrecen-Hungary/protocols/Transformation_of_competent_cells Read more...]<br />
<br />
[[Team:Debrecen-Hungary/protocols#Notebook|[TOP]]]<br />
<br />
=== Mini Prep ===<br />
This protocol is designed for purification of up to 20 μg of high-copy plasmid DNA from 1–5 ml overnight cultures of E. coli in LB (Luria-Bertani) medium [https://2010.igem.org/Team:Debrecen-Hungary/protocols/miniprep Read more...]<br />
<br />
===Midi Prep===<br />
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<object style="height: 170px; width: 270px" ><param name="movie" value="http://www.youtube.com/v/4JyHe5gxihU" ><param name="allowFullScreen" value="true" ><param name="allowScriptAccess" value="always" ><embed src="http://www.youtube.com/v/4JyHe5gxihU" type="application/x-shockwave-flash" allowfullscreen="true" allowScriptAccess="always" width="270" height="170" ></object><br />
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[https://2010.igem.org/Team:Debrecen-Hungary/protocols/midiprep Read more...]<br />
<br />
[[Team:Debrecen-Hungary/protocols#Notebook|[TOP]]]<br />
<br />
== Notebook protocols utilized by the molecular tools subteam ==<br />
<br />
=== Restriction enzyme digestion ===<br />
<br />
BioBrick standard biological parts are flanked by well characterized upstream and downstream sequences which are technically not considered part of the BioBrick part (aka prefix and suffix). These up/down stream segments contain restriction sites for specific restriction enzymes, which allows for the simple creation of larger BioBrick parts by chaining together smaller ones in any desired order.<br />
[https://2010.igem.org/Team:Debrecen-Hungary/protocols/Restriction_biobrick_parts Read more...]<br />
<br />
[[Team:Debrecen-Hungary/protocols#Notebook|[TOP]]]<br />
<br />
=== Cutting gel for PCR product purification ===<br />
<html><br />
<object style="height: 170px; width: 270px" ><param name="movie" value="http://www.youtube.com/v/U74NaSx3rDY" ><param name="allowFullScreen" value="true" ><param name="allowScriptAccess" value="always" ><embed src="http://www.youtube.com/v/U74NaSx3rDY" type="application/x-shockwave-flash" allowfullscreen="true" allowScriptAccess="always" width="270" height="170" ></object><br />
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[https://2010.igem.org/Team:Debrecen-Hungary/protocols/PCR_purification_from_Agarose_Gel Read more...]<br />
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[[Team:Debrecen-Hungary/protocols#Notebook|[TOP]]]<br />
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<br />
== PCR ==<br />
<html><br />
<object style="height: 170px; width: 290px" ><param name="movie" value="http://www.youtube.com/v/vIbKpzlOAXA" ><param name="allowFullScreen" value="true" ><param name="allowScriptAccess" value="always" ><embed src="http://www.youtube.com/v/vIbKpzlOAXA" type="application/x-shockwave-flash" allowfullscreen="true" allowScriptAccess="always" width="290" height="170" ></object><br />
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[https://2010.igem.org/Team:Debrecen-Hungary/protocols/PCR Read more...]<br />
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[[Team:Debrecen-Hungary/protocols#Notebook|[TOP]]]<br />
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=== Gel electrophoresis ===<br />
<html><br />
<object style="height: 170px; width: 270px" ><param name="movie" value="http://www.youtube.com/v/Q-kmMidAo3A" ><param name="allowFullScreen" value="true" ><param name="allowScriptAccess" value="always" ><embed src="http://www.youtube.com/v/Q-kmMidAo3A" type="application/x-shockwave-flash" allowfullscreen="true" allowScriptAccess="always" width="270" height="170" ></object><br />
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[https://2010.igem.org/Team:Debrecen-Hungary/protocols/Gel_electrophoresis Read more...]<br />
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[[Team:Debrecen-Hungary/protocols#Notebook|[TOP]]]<br />
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== Notebook protocols utilized by the tissue culture subteam ==<br />
<br />
=== Cell Passaging ===<br />
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Cell passaging or splitting is a technique that enables an individual to keep cells alive and growing under cultured conditions for extended periods of time. Cells should be passed when they are 90%-100% confluent. You have to do the cell passage on every second to fourth day (i.e. on every Monday, Wednesday and Friday).<br />
After reaching the confluency, the cells do not get enough nutrients and do not have enough place where they can extend. The colour of the medium switches from reddish-pink to orange or yellow which shows acidic metabolic products.<br />
[https://2010.igem.org/Team:Debrecen-Hungary/protocols/Cell_Passaging Read more...]<br />
<br />
[[Team:Debrecen-Hungary/protocols#Notebook|[TOP]]]<br />
<br />
=== Media PEI Preparation ===<br />
Eukaryotic cells, derived from multicellular animal eukaryotes, can be maintained in culturing medias. Aside from temperature and gas mixture, the most commonly varied factor in eucaryotic culture systems is the growth medium. Recipes for growth media can vary in pH, glucose concentration, growth factors, and the presence of other nutrients. The type of the media used depends on the type of the cell line. [https://2010.igem.org/Team:Debrecen-Hungary/protocols/_Media_PEI_Preparation Read more...]<br />
<br />
=== Transfection ===<br />
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The following protocol is used to succesfully introduce foreign plasmid DNA into pluripotent cells through a chemical way, PEI mediated transfection. PEI stands for Poliethilenimine, a cationic polymer which can bind nucleic acids and helps the DNA to get into the cells by receptor-mediated endocytosis.<br />
[https://2010.igem.org/Team:Debrecen-Hungary/protocols/_Transfection Read more...]<br />
<br />
[[Team:Debrecen-Hungary/protocols#Notebook|[TOP]]]<br />
<br />
=== Ligand Treatment ===<br />
Ligand treatment is a procedure when we add the appropriate ligand to the nuclear receptor (NR, transcription factor). The NR got into the cells through a previous transfection step. After treatment the ligand-binded NRs will dimerize and bind to the DNA at specific Nuclear receptor Response Elements and this will promote the gene expression of the downstream gene. In further examinations we detect the expression level of the target gene by Luciferase assay. [https://2010.igem.org/Team:Debrecen-Hungary/protocols/Ligand_Treatment Read more...]<br />
<br />
[[Team:Debrecen-Hungary/protocols#Notebook|[TOP]]]<br />
<br />
== Notebook protocols utilized by the Luciferase subteam ==<br />
<br />
=== Measuring Luciferase activity with the Victor plate reader ===<br />
<br />
he Wallac 1420 VICTOR2 is a multilabel, multitask plate reader designed to support the future demands of industrial and academic laboratories for multiple assay technologies on a single platform. An extended version of the successful Wallac VICTOR multilabel reader, the VICTOR2 allows immediate access to more than 10 counting modes, covering all of the main nonradioactive counting technologies. [https://2010.igem.org/Team:Debrecen-Hungary/protocols/_Enzymatic_activity_with_Victor_machine Read more...]<br />
<br />
[[Team:Debrecen-Hungary/protocols#Notebook|[TOP]]]<br />
<br />
==Online References At Openwetware==<br />
<br />
[https://2010.igem.org/Team:Debrecen-Hungary/protocols/makinglb ''Main article - Making Lurea's Broth'']<br />
<br />
[http://www.openwetware.org/wiki/IGEM:University_of_Debrecen:Transformation_of_competent_cells ''Main article - Transformation of competent cells'']<br />
<br />
[http://openwetware.org/wiki/Editing_IGEM:University_of_Debrecen:_Mini_Prep ''Main article - Mini prep'']<br />
<br />
[http://openwetware.org/wiki/IGEM:University_of_Debrecen:Restriction_biobrick_parts#Notes ''Main article - Restriction enzyme digestion'']<br />
<br />
[http://www.openwetware.org/wiki/IGEM:University_of_Debrecen:PCR_purification_from_Agarose_Gel ''Main article - PCR product purification / Gel purification''] <br><br />
<br />
[http://openwetware.org/wiki/IGEM:University_of_Debrecen:Gel_electrophoresis ''Main article - Gel electrophoresis'']<br />
<br />
[http://www.openwetware.org/wiki/IGEM:University_of_Debrecen:Cell_Passaging ''Main article - Cell passaging'']<br />
<br />
[http://openwetware.org/wiki/IGEM:University_of_Debrecen:_Media_PEI_Preparation ''Main article - Media PEI preparation'']<br />
<br />
[http://www.openwetware.org/wiki/IGEM:University_of_Debrecen:_Transfection ''Main article - Transfection'']<br />
<br />
[http://openwetware.org/wiki/IGEM:University_of_Debrecen:Ligand_Treatment ''Main article - Ligand treatment''] <br />
<br />
[http://openwetware.org/wiki/IGEM:University_of_Debrecen:_Enzymatic_activity_with_Victor_machine ''Main article - Measuring Luciferase activity with the Victor plate reader'']<br />
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</html></div>Liorhttp://2010.igem.org/Team:Debrecen-Hungary/protocols/_TransfectionTeam:Debrecen-Hungary/protocols/ Transfection2010-10-26T21:48:44Z<p>Lior: </p>
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<div>{{Template:Debrecen-Hungary_secret}}<br />
<br />
Transfection protocol with PEI nuclear receptor construct<br />
(for COS1 cells)<br />
<br />
==Scientific Background==<br />
<br />
The following protocol is used to succesfully introduce foreign plasmid DNA into pluripotent cells through a chemical way, PEI mediated transfection. PEI stands for Poliethilenimine, a cationic polymer which can bind nucleic acids and helps the DNA to get into the cells by receptor-mediated endocytosis.<br />
<br />
==Overview==<br />
<br />
Performing the protocol from beginning to end takes approximately one and a half day and it is divided into two major procedures:<br />
<br />
1.Plating COS1 cells into 48 well plates in order to get 60% confluency of adherent cells for transfection<br />
<br />
The goal is to get 60% confluency of adhaerent cells on a 48 well plate before transfect them<br />
<br />
2. The PEI mediated transfection itself<br />
<br />
The goal is to introduce foreign plasmid DNA successfully into COS1 cells 24 hours after the plating<br />
<br />
==Materials==<br />
<br />
<h5>I.Plating COS1 cells:</h5><br />
<br />
48 well plates <br />
<br />
DMEM Medium containing 10% FBS <br />
<br />
Trypsin-EDTA <br />
<br />
1x PBS <br />
<br />
Brüker-chamber<br />
<br />
centrifuge tubes<br />
<br />
serological pipettes<br />
<br />
pipettes and tips<br />
<br />
vacuum aspirator<br />
<br />
Pasteur pipettes <br />
<br />
Sterile laminar air flow box<br />
<br />
incubators <br />
<br />
<br />
<h5>II. PEI mediated transfection</h5><br />
<br />
Plated cells <br />
<br />
Serum Free DMEM Medium <br />
<br />
sterile 100 mM PEI solution<br />
<br />
sterile 150 mM NaCl solution<br />
<br />
TE –buffer (filtered) <br />
<br />
Plasmids: Beta-Gal (normalizer), Luciferase (tracer), Receptor1, Receptor2, VDR-1 (MOCK, negative controll) - their concentration has been measured previously<br />
<br />
Eppendorf tubes<br />
<br />
centrifuge tubes<br />
<br />
serological pipettes<br />
<br />
pipettes and tips<br />
<br />
vacuum aspirator <br />
<br />
Pasteur pipettes <br />
<br />
Sterile laminar air flow box<br />
<br />
incubators<br />
<br />
<br />
==Procedure==<br />
<br />
<h5>I. Plating COS1 cells:</h5><br><br />
<br />
1. Prepare the sterile box, and prewarm the medium, trypsin-EDTA and PBS up to 37°C<br><br><br />
<br />
2. Get the cells to the sterile box in a closed cell culturing flask or Petri dish. <br>You should open it only under the sterile box.<Br><br><br />
<br />
3. Gently remove used medium from the cells using vacuum aspirator with Pasteur pipette<br><br><br />
<br />
4. Wash the cells with 2-3 ml PBS, then remove it gently by vacuum aspirator with Pasteur pipette<Br><br><br />
<br />
5. Incubate the Cos1 cells for 3-5 mins with 2 ml of trypsin-EDTA at 370C, <br>then check them under phase-contrast microscope whether they are detached from the surface<br><br><br />
<br />
6. Add 4 ml medium(you can change the dilution level depending on the cell number, in order to be able to count the cells easier) to the trypsinised cells,<br> re-suspend them and check them under phase-contrast microscope to make sure that you got individual cells<br><br><br />
<br />
7. Prepare the Bürker-chamber and do a cell count<br><br><br />
<br />
8.To reach 60% confluency the day after plating, we put 21.000 cells into each well<br><br><br />
<br />
For a 48 well plate, if we calculate with 60 wells, we put into a 50 ml centrifuge tube:<br />
- 21000x60= 1.320.000 cells [ cellnumber in 1 ml / 1.320.000 ML cell susp. ] , and if the total volume of the wells are 200 ul, <br />
- we fill the cell suspension up to 200x60 ul= 12 ml with 10% FBS DMEM<br><br><br />
<br />
9. By using a repeating pipet, put 200 ul from this suspension into each well, swirl the plate<br><br><br />
<br />
10. Incubate the cells for 1 day at 37°C, 5% CO2<br><br><br />
<br />
<br />
<h5>II.PEI mediated transfection</h5><br><br />
<br />
1.Prepare the sterile box, and prewarm the medium up to 37°C<Br><br><br />
<br />
2.Change the medium at least 1 hour before transfection to Serum Free DMEM.<br> (gently remove used medium from the cells using vacuum aspirator with Pasteur pipette, then pipette 200 µl Serum Free DMEM<br><br><br />
<br />
3. Mix gently plasmid solutions (Do not vortex!)<br><br><br />
<br />
4. Dilute plasmids to 0,1 µg/µl cc. with TE-buffer in sterile Eppendorf tubes<br><br><br />
<br />
5. Prepare DNA mixes:<br />
<br />
T A B L E I N S E R T R E Q U I R E D<br><br />
Prepare the mastermix: 129,6 ul bGAL, 165,6 ul Luc<br><br />
<br />
Put 66-66 ul Mastermix 1 into four Eppendorf tubes. The plasmids of special receptors have to be added to each Eppendorf tubes (7 µl from each)<Br><br />
<br />
in the case of PPAR gamma construct transfection:<br><br />
B-gal: 180 ng/well<br><br />
Luc: 230 ng/well<br><br />
GAL4-PPARg: 120 ng/well<br><br />
as we need 25 µl 150 mM NaCl solution/well,<br> we put 16x25=400 ul into each DNA mix Eppendor tube, then vortex briefly<br><br><br />
<br />
6. Prepare PEI mix in 4 pieces of eppendorf tubes:<br />
<br />
A. Per 1 well:<br><br />
PEI μL 1.3<Br><br />
NaCL μL 25<br><br />
<br />
B. Per 16 well:<br><br />
PEI μL 20.8<br><br />
NaCL μL 400<br><br />
<br />
vortex the PEI- NaCl mixes in the Eppendorf tubes briefly.<br />
<br />
<br />
<br />
7. Add PEI mixes to DNA mixes dopwise , then vortex briefly. (Do not add DNA mixes to PEI mix!)<br><br><br />
<br />
8. Incubate transfection mixes for 20 mins at room temperature<Br><br><br />
<br />
9. Add total transf.mix.volume in one epp.tube/16 µl transfection mix to each well in drops, then swirl gently the plates<Br><br><br />
<br />
48-well plates, 8 rows, 6 columns:<br />
<br />
1st row: (VDR-)x2<br />
<br />
2nd row: Rec1+ VDR- <br />
<br />
3rd row: Rec2+VDR-<br />
<br />
4th row: Rec1+Rec2<br />
<br />
5th row:(VDR-)x2<br />
<br />
6th row:Rec1+ VDR-<br />
<br />
7th row:Rec2+VDR-<br />
<br />
8th row:Rec1+Rec2<br />
<br />
<br />
10. Incubate the cells for 5-6 hours at 370C, 5% CO2<br><br><br><br><br><br />
<br />
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==Notes & troubleshooting==<br />
==References==<br />
1. Horbinski C, Stachowiak MK, Higgins D, Finnegan SG. Polyethyleneimine-mediated transfection of cultured postmitotic neurons from rat sympathetic ganglia and adult human retina. .[http://www.biomedcentral.com/1471-2202/2/2 BMC Neurosci. 2001;2:2].<br />
<br />
2. Pollard H, Remy JS, Loussouarn G, Demolombe S, Behr JP, Escande D: Polyethylenimine but not cationic lipids promotes transgene delivery to the nucleus in mammalian cells.[http://www.jbc.org/content/273/13/7507.long J Biol Chem 1998], 273:7507-7511<br />
<br />
==Links==<br />
<br />
[http://www.youtube.com/user/debrecenigem2010#p/u/13/r8DlyOCRJRs Video I]<br />
<br />
[http://www.youtube.com/user/debrecenigem2010#p/u/12/IgRxD-Tf5Eo Video II]</div>Liorhttp://2010.igem.org/Team:Debrecen-Hungary/protocols/_TransfectionTeam:Debrecen-Hungary/protocols/ Transfection2010-10-26T21:47:00Z<p>Lior: /* Procedure */</p>
<hr />
<div>{{Template:Debrecen-Hungary_secret}}<br />
<br />
Transfection protocol with PEI nuclear receptor construct<br />
(for COS1 cells)<br />
<br />
==Scientific Background==<br />
<br />
The following protocol is used to succesfully introduce foreign plasmid DNA into pluripotent cells through a chemical way, PEI mediated transfection. PEI stands for Poliethilenimine, a cationic polymer which can bind nucleic acids and helps the DNA to get into the cells by receptor-mediated endocytosis.<br />
<br />
==Overview==<br />
<br />
Performing the protocol from beginning to end takes approximately one and a half day and it is divided into two major procedures:<br />
<br />
1.Plating COS1 cells into 48 well plates in order to get 60% confluency of adherent cells for transfection<br />
<br />
The goal is to get 60% confluency of adhaerent cells on a 48 well plate before transfect them<br />
<br />
2. The PEI mediated transfection itself<br />
<br />
The goal is to introduce foreign plasmid DNA successfully into COS1 cells 24 hours after the plating<br />
<br />
==Materials==<br />
<br />
<h5>I.Plating COS1 cells:</h5><br />
<br />
48 well plates <br />
<br />
DMEM Medium containing 10% FBS <br />
<br />
Trypsin-EDTA <br />
<br />
1x PBS <br />
<br />
Brüker-chamber<br />
<br />
centrifuge tubes<br />
<br />
serological pipettes<br />
<br />
pipettes and tips<br />
<br />
vacuum aspirator<br />
<br />
Pasteur pipettes <br />
<br />
Sterile laminar air flow box<br />
<br />
incubators <br />
<br />
<br />
<h5>II. PEI mediated transfection</h5><br />
<br />
Plated cells <br />
<br />
Serum Free DMEM Medium <br />
<br />
sterile 100 mM PEI solution<br />
<br />
sterile 150 mM NaCl solution<br />
<br />
TE –buffer (filtered) <br />
<br />
Plasmids: Beta-Gal (normalizer), Luciferase (tracer), Receptor1, Receptor2, VDR-1 (MOCK, negative controll) - their concentration has been measured previously<br />
<br />
Eppendorf tubes<br />
<br />
centrifuge tubes<br />
<br />
serological pipettes<br />
<br />
pipettes and tips<br />
<br />
vacuum aspirator <br />
<br />
Pasteur pipettes <br />
<br />
Sterile laminar air flow box<br />
<br />
incubators<br />
<br />
<br />
==Procedure==<br />
<br />
<h5>I. Plating COS1 cells:</h5><br><br />
<br />
1. Prepare the sterile box, and prewarm the medium, trypsin-EDTA and PBS up to 37°C<br><br><br />
<br />
2. Get the cells to the sterile box in a closed cell culturing flask or Petri dish. <br>You should open it only under the sterile box.<Br><br><br />
<br />
3. Gently remove used medium from the cells using vacuum aspirator with Pasteur pipette<br><br><br />
<br />
4. Wash the cells with 2-3 ml PBS, then remove it gently by vacuum aspirator with Pasteur pipette<Br><br><br />
<br />
5. Incubate the Cos1 cells for 3-5 mins with 2 ml of trypsin-EDTA at 370C, <br>then check them under phase-contrast microscope whether they are detached from the surface<br><br><br />
<br />
6. Add 4 ml medium(you can change the dilution level depending on the cell number, in order to be able to count the cells easier) to the trypsinised cells,<br> re-suspend them and check them under phase-contrast microscope to make sure that you got individual cells<br><br><br />
<br />
7. Prepare the Bürker-chamber and do a cell count<br><br><br />
<br />
8.To reach 60% confluency the day after plating, we put 21.000 cells into each well<br><br><br />
<br />
For a 48 well plate, if we calculate with 60 wells, we put into a 50 ml centrifuge tube:<br />
- 21000x60= 1.320.000 cells [ cellnumber in 1 ml / 1.320.000 ML cell susp. ] , and if the total volume of the wells are 200 ul, <br />
- we fill the cell suspension up to 200x60 ul= 12 ml with 10% FBS DMEM<br><br><br />
<br />
9. By using a repeating pipet, put 200 ul from this suspension into each well, swirl the plate<br><br><br />
<br />
10. Incubate the cells for 1 day at 37°C, 5% CO2<br><br><br />
<br />
<br />
<h5>II.PEI mediated transfection</h5><br><br />
<br />
1.Prepare the sterile box, and prewarm the medium up to 37°C<Br><br><br />
<br />
2.Change the medium at least 1 hour before transfection to Serum Free DMEM.<br> (gently remove used medium from the cells using vacuum aspirator with Pasteur pipette, then pipette 200 µl Serum Free DMEM<br><br><br />
<br />
3. Mix gently plasmid solutions (Do not vortex!)<br><br><br />
<br />
4. Dilute plasmids to 0,1 µg/µl cc. with TE-buffer in sterile Eppendorf tubes<br><br><br />
<br />
5. Prepare DNA mixes:<br />
<br />
T A B L E I N S E R T R E Q U I R E D<br><br />
Prepare the mastermix: 129,6 ul bGAL, 165,6 ul Luc<br><br />
<br />
Put 66-66 ul Mastermix 1 into four Eppendorf tubes. The plasmids of special receptors have to be added to each Eppendorf tubes (7 µl from each)<Br><br />
<br />
in the case of PPAR gamma construct transfection:<br><br />
B-gal: 180 ng/well<br><br />
Luc: 230 ng/well<br><br />
GAL4-PPARg: 120 ng/well<br><br />
as we need 25 µl 150 mM NaCl solution/well,<br> we put 16x25=400 ul into each DNA mix Eppendor tube, then vortex briefly<br><br><br />
<br />
6. Prepare PEI mix in 4 pieces of eppendorf tubes:<br />
<br />
A. Per 1 well:<br><br />
PEI μL 1.3<Br><br />
NaCL μL 25<br><br />
<br />
B. Per 16 well:<br><br />
PEI μL 20.8<br><br />
NaCL μL 400<br><br />
<br />
vortex the PEI- NaCl mixes in the Eppendorf tubes briefly.<br />
<br />
<br />
<br />
7. Add PEI mixes to DNA mixes dopwise , then vortex briefly. (Do not add DNA mixes to PEI mix!)<br><br><br />
<br />
8. Incubate transfection mixes for 20 mins at room temperature<Br><br><br />
<br />
9. Add total transf.mix.volume in one epp.tube/16 µl transfection mix to each well in drops, then swirl gently the plates<Br><br><br />
<br />
48-well plates, 8 rows, 6 columns:<br />
<br />
1st row: (VDR-)x2<br />
<br />
2nd row: Rec1+ VDR- <br />
<br />
3rd row: Rec2+VDR-<br />
<br />
4th row: Rec1+Rec2<br />
<br />
5th row:(VDR-)x2<br />
<br />
6th row:Rec1+ VDR-<br />
<br />
7th row:Rec2+VDR-<br />
<br />
8th row:Rec1+Rec2<br />
<br />
<br />
10. Incubate the cells for 5-6 hours at 370C, 5% CO2<br><br><br />
<br />
<html><br />
<object style="height: 190px; width: 290px" ><param name="movie" value="http://www.youtube.com/v/r8DlyOCRJRs" ><param name="allowFullScreen" value="true" ><param name="allowScriptAccess" value="always" ><embed src="http://www.youtube.com/v/r8DlyOCRJRs" type="application/x-shockwave-flash" allowfullscreen="true" allowScriptAccess="always" width="290" height="190"></object><br />
</html><br />
&nbsp;&nbsp;<br />
<html><br />
<object style="height: 190px; width: 290px" ><param name="movie" value="http://www.youtube.com/v/IgRxD-Tf5Eo" ><param name="allowFullScreen" value="true" ><param name="allowScriptAccess" value="always" ><embed src="http://www.youtube.com/v/IgRxD-Tf5Eo" type="application/x-shockwave-flash" allowfullscreen="true" allowScriptAccess="always" width="290" height="190" ></object><br />
</html><br />
<br />
==Notes & troubleshooting==<br />
==References==<br />
1. Horbinski C, Stachowiak MK, Higgins D, Finnegan SG. Polyethyleneimine-mediated transfection of cultured postmitotic neurons from rat sympathetic ganglia and adult human retina. .[http://www.biomedcentral.com/1471-2202/2/2 BMC Neurosci. 2001;2:2].<br />
<br />
2. Pollard H, Remy JS, Loussouarn G, Demolombe S, Behr JP, Escande D: Polyethylenimine but not cationic lipids promotes transgene delivery to the nucleus in mammalian cells.[http://www.jbc.org/content/273/13/7507.long J Biol Chem 1998], 273:7507-7511<br />
<br />
==Links==</div>Liorhttp://2010.igem.org/Team:Debrecen-Hungary/protocols/PCRTeam:Debrecen-Hungary/protocols/PCR2010-10-26T21:07:42Z<p>Lior: /* Links */</p>
<hr />
<div>{{Template:Debrecen-Hungary_secret}}<br />
<br />
<br />
<br />
<br />
==Overview==<br />
<br />
PCR is an acronym for polymerase chain reaction. It is a method for amplifying DNA in vitro.<br />
<br />
==Reagents==<br />
• Expand High Fidelity Buffer with MgCl2<br />
<br />
• PCR grade nucleotide mix MgCl 2<br />
<br />
• Upstream primer<br />
<br />
• Downstream primer<br />
<br />
• Template DNA<br />
<br />
• RNase free water <br />
<br />
• Expand High Fidelity Enzyme Mix<br />
==Equipment==<br />
• Thermocycler<br />
<br />
• Reaction tubes<br />
<br />
==Step==<br />
'''Prepare Master mix 1'''<br />
<br />
1. Add RNase free water to reach the final volume of 25 microliter<br />
<div style="float: right;"><html><br />
<object style="height: 170px; width: 290px" ><param name="movie" value="http://www.youtube.com/v/vIbKpzlOAXA" ><param name="allowFullScreen" value="true" ><param name="allowScriptAccess" value="always" ><embed src="http://www.youtube.com/v/vIbKpzlOAXA" type="application/x-shockwave-flash" allowfullscreen="true" allowScriptAccess="always" width="290" height="170" ></object><br />
</html></div><br><br />
<br />
<br />
2. Add 1 microliter of PCR grade nucleotide mix <br><br><br><br />
<br />
3. Add downstream primer to reach the final concentration of 300 nanomolar<Br><br><br />
<br />
<br />
4. Add upstream primer in the same way.<br />
<div style="float: right;"><html><br />
<object style="height: 170px; width: 290px" ><param name="movie" value="http://www.youtube.com/v/j-hmoyMSc2Y" ><param name="allowFullScreen" value="true" ><param name="allowScriptAccess" value="always" ><embed src="http://www.youtube.com/v/j-hmoyMSc2Y" type="application/x-shockwave-flash" allowfullscreen="true" allowScriptAccess="always" width="290" height="170" ></object><br />
</html></div><br><br />
<br />
<br />
5. Add Template DNA which contains 10 to 250 nanogram complex DNA <br><br><br><br />
<br />
'''Prepare Master mix 2'''<Br><br><br><br />
<br />
6. Add 19.25 microliter of RNase free water<Br><br />
<div style="float: right;"><html><br />
<object style="height: 170px; width: 290px" ><param name="movie" value="http://www.youtube.com/v/8_cafnNEqhU" ><param name="allowFullScreen" value="true" ><param name="allowScriptAccess" value="always" ><embed src="http://www.youtube.com/v/8_cafnNEqhU" type="application/x-shockwave-flash" allowfullscreen="true" allowScriptAccess="always" width="290" height="170" ></object><br />
</html></div><br><br><br />
<br />
7. Add 5 microliter of Expand High Fidelity Buffer with MgCl2<Br><br><br><br />
<br />
8. Add 0.75 of Expand High Fidelity Enzyme Mix<Br><br><Br><br />
<br />
9. Vortex the components in both Master mixes<Br><br />
<div style="float: right;"><html><br />
<object style="height: 170px; width: 290px" ><param name="movie" value="http://www.youtube.com/v/9fUZtcbOvqs" ><param name="allowFullScreen" value="true" ><param name="allowScriptAccess" value="always" ><embed src="http://www.youtube.com/v/9fUZtcbOvqs" type="application/x-shockwave-flash" allowfullscreen="true" allowScriptAccess="always" width="290" height="170" ></object><br />
</html></div><br><br />
<br />
<br />
10. Prepare the PCR tubes<Br><br><br><br />
<br />
11. Divide the mix1 into the tubes in a desired volume<Br><br><br><br />
<br />
12. Then add the mix2 into the PCR tubes which contain mix 1<Br><br />
<div style="float: right;"><html><br />
<object style="height: 170px; width: 290px" ><param name="movie" value="http://www.youtube.com/v/nDOxB_-aST0" ><param name="allowFullScreen" value="true" ><param name="allowScriptAccess" value="always" ><embed src="http://www.youtube.com/v/nDOxB_-aST0" type="application/x-shockwave-flash" allowfullscreen="true" allowScriptAccess="always" width="290" height="170" ></object><br />
</html></div><br><br><br />
<br />
13. Set up the Thermocycler<br><br><br><br />
<br />
14. Place the PCR tubes into the thermocycler.<br><br />
<br />
<br><br><br><br><br />
<br />
==An example program==<br />
Program a standard thermocycler to run the reaction using the following parameters:<br />
Initial denaturation<br />
• Denature: 96°C, 5 mins<br />
This denatures any double stranded DNA.<br />
Thermocycling<br><br />
• No. of cycles: 25<br><br />
• Denature: 96°C, 1 min<br><br />
• Anneal: 55°C, 30 secs<br><br />
• Elongate: 72°C, 1 min<Br><br />
Termination<br><br />
• Elongate: 72°C, 5 mins<br><br />
• Hold: 4°C, until removed from machine<br><br />
<br />
<br />
<br />
==References==<br />
<br />
1. Arezi B, Xing W, Sorge JA, and Hogrefe HH. Amplification efficiency of thermostable DNA polymerases. Anal Biochem 2003 Oct 15; 321(2) 226-35. pmid:14511688. PubMed HubMed [Arezi-AnalBiochem-2003]<br />
<br />
2. Saiki RK, Scharf S, Faloona F, Mullis KB, Horn GT, Erlich HA, and Arnheim N. Enzymatic amplification of beta-globin genomic sequences and restriction site analysis for diagnosis of sickle cell anemia.Science 1985 Dec 20; 230(4732) 1350-4. pmid:2999980. PubMed HubMed [Saiki-Science-1985]<br />
original paper on PCR<br />
<br />
3. Mullis K, Faloona F, Scharf S, Saiki R, Horn G, and Erlich H. Specific enzymatic amplification of DNA in vitro: the polymerase chain reaction. Cold Spring Harb Symp Quant Biol 1986; 51 Pt 1 263-73.pmid:3472723. PubMed HubMed [Mullis-1986]<br />
first public presentation on PCR<br />
<br />
<br />
==Links==<br />
<br />
[http://www.youtube.com/user/debrecenigem2010#p/u/11/vIbKpzlOAXA Video I & II]<br />
<br />
[http://www.youtube.com/user/debrecenigem2010#p/u/10/j-hmoyMSc2Y Video III]<br />
<br />
[http://www.youtube.com/user/debrecenigem2010#p/u/9/8_cafnNEqhU Video IV]<br />
<br />
[http://www.youtube.com/user/debrecenigem2010#p/u/8/9fUZtcbOvqs Video V]<br />
<br />
[http://www.youtube.com/user/debrecenigem2010#p/u/7/nDOxB_-aST0 Video VI]</div>Liorhttp://2010.igem.org/Team:Debrecen-Hungary/protocols/PCRTeam:Debrecen-Hungary/protocols/PCR2010-10-26T21:07:21Z<p>Lior: </p>
<hr />
<div>{{Template:Debrecen-Hungary_secret}}<br />
<br />
<br />
<br />
<br />
==Overview==<br />
<br />
PCR is an acronym for polymerase chain reaction. It is a method for amplifying DNA in vitro.<br />
<br />
==Reagents==<br />
• Expand High Fidelity Buffer with MgCl2<br />
<br />
• PCR grade nucleotide mix MgCl 2<br />
<br />
• Upstream primer<br />
<br />
• Downstream primer<br />
<br />
• Template DNA<br />
<br />
• RNase free water <br />
<br />
• Expand High Fidelity Enzyme Mix<br />
==Equipment==<br />
• Thermocycler<br />
<br />
• Reaction tubes<br />
<br />
==Step==<br />
'''Prepare Master mix 1'''<br />
<br />
1. Add RNase free water to reach the final volume of 25 microliter<br />
<div style="float: right;"><html><br />
<object style="height: 170px; width: 290px" ><param name="movie" value="http://www.youtube.com/v/vIbKpzlOAXA" ><param name="allowFullScreen" value="true" ><param name="allowScriptAccess" value="always" ><embed src="http://www.youtube.com/v/vIbKpzlOAXA" type="application/x-shockwave-flash" allowfullscreen="true" allowScriptAccess="always" width="290" height="170" ></object><br />
</html></div><br><br />
<br />
<br />
2. Add 1 microliter of PCR grade nucleotide mix <br><br><br><br />
<br />
3. Add downstream primer to reach the final concentration of 300 nanomolar<Br><br><br />
<br />
<br />
4. Add upstream primer in the same way.<br />
<div style="float: right;"><html><br />
<object style="height: 170px; width: 290px" ><param name="movie" value="http://www.youtube.com/v/j-hmoyMSc2Y" ><param name="allowFullScreen" value="true" ><param name="allowScriptAccess" value="always" ><embed src="http://www.youtube.com/v/j-hmoyMSc2Y" type="application/x-shockwave-flash" allowfullscreen="true" allowScriptAccess="always" width="290" height="170" ></object><br />
</html></div><br><br />
<br />
<br />
5. Add Template DNA which contains 10 to 250 nanogram complex DNA <br><br><br><br />
<br />
'''Prepare Master mix 2'''<Br><br><br><br />
<br />
6. Add 19.25 microliter of RNase free water<Br><br />
<div style="float: right;"><html><br />
<object style="height: 170px; width: 290px" ><param name="movie" value="http://www.youtube.com/v/8_cafnNEqhU" ><param name="allowFullScreen" value="true" ><param name="allowScriptAccess" value="always" ><embed src="http://www.youtube.com/v/8_cafnNEqhU" type="application/x-shockwave-flash" allowfullscreen="true" allowScriptAccess="always" width="290" height="170" ></object><br />
</html></div><br><br><br />
<br />
7. Add 5 microliter of Expand High Fidelity Buffer with MgCl2<Br><br><br><br />
<br />
8. Add 0.75 of Expand High Fidelity Enzyme Mix<Br><br><Br><br />
<br />
9. Vortex the components in both Master mixes<Br><br />
<div style="float: right;"><html><br />
<object style="height: 170px; width: 290px" ><param name="movie" value="http://www.youtube.com/v/9fUZtcbOvqs" ><param name="allowFullScreen" value="true" ><param name="allowScriptAccess" value="always" ><embed src="http://www.youtube.com/v/9fUZtcbOvqs" type="application/x-shockwave-flash" allowfullscreen="true" allowScriptAccess="always" width="290" height="170" ></object><br />
</html></div><br><br />
<br />
<br />
10. Prepare the PCR tubes<Br><br><br><br />
<br />
11. Divide the mix1 into the tubes in a desired volume<Br><br><br><br />
<br />
12. Then add the mix2 into the PCR tubes which contain mix 1<Br><br />
<div style="float: right;"><html><br />
<object style="height: 170px; width: 290px" ><param name="movie" value="http://www.youtube.com/v/nDOxB_-aST0" ><param name="allowFullScreen" value="true" ><param name="allowScriptAccess" value="always" ><embed src="http://www.youtube.com/v/nDOxB_-aST0" type="application/x-shockwave-flash" allowfullscreen="true" allowScriptAccess="always" width="290" height="170" ></object><br />
</html></div><br><br><br />
<br />
13. Set up the Thermocycler<br><br><br><br />
<br />
14. Place the PCR tubes into the thermocycler.<br><br />
<br />
<br><br><br><br><br />
<br />
==An example program==<br />
Program a standard thermocycler to run the reaction using the following parameters:<br />
Initial denaturation<br />
• Denature: 96°C, 5 mins<br />
This denatures any double stranded DNA.<br />
Thermocycling<br><br />
• No. of cycles: 25<br><br />
• Denature: 96°C, 1 min<br><br />
• Anneal: 55°C, 30 secs<br><br />
• Elongate: 72°C, 1 min<Br><br />
Termination<br><br />
• Elongate: 72°C, 5 mins<br><br />
• Hold: 4°C, until removed from machine<br><br />
<br />
<br />
<br />
==References==<br />
<br />
1. Arezi B, Xing W, Sorge JA, and Hogrefe HH. Amplification efficiency of thermostable DNA polymerases. Anal Biochem 2003 Oct 15; 321(2) 226-35. pmid:14511688. PubMed HubMed [Arezi-AnalBiochem-2003]<br />
<br />
2. Saiki RK, Scharf S, Faloona F, Mullis KB, Horn GT, Erlich HA, and Arnheim N. Enzymatic amplification of beta-globin genomic sequences and restriction site analysis for diagnosis of sickle cell anemia.Science 1985 Dec 20; 230(4732) 1350-4. pmid:2999980. PubMed HubMed [Saiki-Science-1985]<br />
original paper on PCR<br />
<br />
3. Mullis K, Faloona F, Scharf S, Saiki R, Horn G, and Erlich H. Specific enzymatic amplification of DNA in vitro: the polymerase chain reaction. Cold Spring Harb Symp Quant Biol 1986; 51 Pt 1 263-73.pmid:3472723. PubMed HubMed [Mullis-1986]<br />
first public presentation on PCR<br />
<br />
<br />
==Links==<br />
<br />
[http://www.youtube.com/user/debrecenigem2010#p/u/11/vIbKpzlOAXA Video I & II]<br />
<br />
[http://www.youtube.com/user/debrecenigem2010#p/u/10/j-hmoyMSc2Y Video III]<br />
<br />
[http://www.youtube.com/user/debrecenigem2010#p/u/9/8_cafnNEqhU Video IV]<br />
<br />
[http://www.youtube.com/user/debrecenigem2010#p/u/8/9fUZtcbOvqs Video V]<br />
<br />
[http://www.youtube.com/user/debrecenigem2010#p/u/7/nDOxB_-aST0 Video IV]</div>Liorhttp://2010.igem.org/Team:Debrecen-Hungary/protocols/PCRTeam:Debrecen-Hungary/protocols/PCR2010-10-26T21:00:51Z<p>Lior: </p>
<hr />
<div>{{Template:Debrecen-Hungary_secret}}<br />
<br />
<br />
<br />
<br />
==Overview==<br />
<br />
PCR is an acronym for polymerase chain reaction. It is a method for amplifying DNA in vitro.<br />
<br />
==Reagents==<br />
• Expand High Fidelity Buffer with MgCl2<br />
<br />
• PCR grade nucleotide mix MgCl 2<br />
<br />
• Upstream primer<br />
<br />
• Downstream primer<br />
<br />
• Template DNA<br />
<br />
• RNase free water <br />
<br />
• Expand High Fidelity Enzyme Mix<br />
==Equipment==<br />
• Thermocycler<br />
<br />
• Reaction tubes<br />
<br />
==Step==<br />
'''Prepare Master mix 1'''<br />
<br />
1. Add RNase free water to reach the final volume of 25 microliter<br />
<div style="float: right;"><html><br />
<object style="height: 170px; width: 290px" ><param name="movie" value="http://www.youtube.com/v/vIbKpzlOAXA" ><param name="allowFullScreen" value="true" ><param name="allowScriptAccess" value="always" ><embed src="http://www.youtube.com/v/vIbKpzlOAXA" type="application/x-shockwave-flash" allowfullscreen="true" allowScriptAccess="always" width="290" height="170" ></object><br />
</html></div><br><br />
<br />
<br />
2. Add 1 microliter of PCR grade nucleotide mix <br><br><br><br />
<br />
3. Add downstream primer to reach the final concentration of 300 nanomolar<Br><br><br />
<br />
<br />
4. Add upstream primer in the same way.<br />
<div style="float: right;"><html><br />
<object style="height: 170px; width: 290px" ><param name="movie" value="http://www.youtube.com/v/j-hmoyMSc2Y" ><param name="allowFullScreen" value="true" ><param name="allowScriptAccess" value="always" ><embed src="http://www.youtube.com/v/j-hmoyMSc2Y" type="application/x-shockwave-flash" allowfullscreen="true" allowScriptAccess="always" width="290" height="170" ></object><br />
</html></div><br><br />
<br />
<br />
5. Add Template DNA which contains 10 to 250 nanogram complex DNA <br><br><br><br />
<br />
'''Prepare Master mix 2'''<Br><br><br><br />
<br />
6. Add 19.25 microliter of RNase free water<Br><br />
<div style="float: right;"><html><br />
<object style="height: 170px; width: 290px" ><param name="movie" value="http://www.youtube.com/v/8_cafnNEqhU" ><param name="allowFullScreen" value="true" ><param name="allowScriptAccess" value="always" ><embed src="http://www.youtube.com/v/8_cafnNEqhU" type="application/x-shockwave-flash" allowfullscreen="true" allowScriptAccess="always" width="290" height="170" ></object><br />
</html></div><br><br><br />
<br />
7. Add 5 microliter of Expand High Fidelity Buffer with MgCl2<Br><br><br><br />
<br />
8. Add 0.75 of Expand High Fidelity Enzyme Mix<Br><br><Br><br />
<br />
9. Vortex the components in both Master mixes<Br><br />
<div style="float: right;"><html><br />
<object style="height: 170px; width: 290px" ><param name="movie" value="http://www.youtube.com/v/9fUZtcbOvqs" ><param name="allowFullScreen" value="true" ><param name="allowScriptAccess" value="always" ><embed src="http://www.youtube.com/v/9fUZtcbOvqs" type="application/x-shockwave-flash" allowfullscreen="true" allowScriptAccess="always" width="290" height="170" ></object><br />
</html></div><br><br />
<br />
<br />
10. Prepare the PCR tubes<Br><br><br><br />
<br />
11. Divide the mix1 into the tubes in a desired volume<Br><br><br><br />
<br />
12. Then add the mix2 into the PCR tubes which contain mix 1<Br><br />
<div style="float: right;"><html><br />
<object style="height: 170px; width: 290px" ><param name="movie" value="http://www.youtube.com/v/nDOxB_-aST0" ><param name="allowFullScreen" value="true" ><param name="allowScriptAccess" value="always" ><embed src="http://www.youtube.com/v/nDOxB_-aST0" type="application/x-shockwave-flash" allowfullscreen="true" allowScriptAccess="always" width="290" height="170" ></object><br />
</html></div><br><br><br />
<br />
13. Set up the Thermocycler<br><br><br><br />
<br />
14. Place the PCR tubes into the thermocycler.<br><br />
<br />
<br><br><br><br><br />
<br />
==An example program==<br />
Program a standard thermocycler to run the reaction using the following parameters:<br />
Initial denaturation<br />
• Denature: 96°C, 5 mins<br />
This denatures any double stranded DNA.<br />
Thermocycling<br><br />
• No. of cycles: 25<br><br />
• Denature: 96°C, 1 min<br><br />
• Anneal: 55°C, 30 secs<br><br />
• Elongate: 72°C, 1 min<Br><br />
Termination<br><br />
• Elongate: 72°C, 5 mins<br><br />
• Hold: 4°C, until removed from machine<br><br />
<br />
<br />
<br />
==References==<br />
<br />
1. Arezi B, Xing W, Sorge JA, and Hogrefe HH. Amplification efficiency of thermostable DNA polymerases. Anal Biochem 2003 Oct 15; 321(2) 226-35. pmid:14511688. PubMed HubMed [Arezi-AnalBiochem-2003]<br />
<br />
2. Saiki RK, Scharf S, Faloona F, Mullis KB, Horn GT, Erlich HA, and Arnheim N. Enzymatic amplification of beta-globin genomic sequences and restriction site analysis for diagnosis of sickle cell anemia.Science 1985 Dec 20; 230(4732) 1350-4. pmid:2999980. PubMed HubMed [Saiki-Science-1985]<br />
original paper on PCR<br />
<br />
3. Mullis K, Faloona F, Scharf S, Saiki R, Horn G, and Erlich H. Specific enzymatic amplification of DNA in vitro: the polymerase chain reaction. Cold Spring Harb Symp Quant Biol 1986; 51 Pt 1 263-73.pmid:3472723. PubMed HubMed [Mullis-1986]<br />
first public presentation on PCR</div>Liorhttp://2010.igem.org/Team:Debrecen-Hungary/protocols/PCRTeam:Debrecen-Hungary/protocols/PCR2010-10-26T20:57:21Z<p>Lior: /* Step */</p>
<hr />
<div>{{Template:Debrecen-Hungary_secret}}<br />
<br />
<br />
<br />
<br />
==Overview==<br />
<br />
PCR is an acronym for polymerase chain reaction. It is a method for amplifying DNA in vitro.<br />
<br />
==Reagents==<br />
• Expand High Fidelity Buffer with MgCl2<br />
<br />
• PCR grade nucleotide mix MgCl 2<br />
<br />
• Upstream primer<br />
<br />
• Downstream primer<br />
<br />
• Template DNA<br />
<br />
• RNase free water <br />
<br />
• Expand High Fidelity Enzyme Mix<br />
==Equipment==<br />
• Thermocycler<br />
<br />
• Reaction tubes<br />
<br />
==Step==<br />
'''Prepare Master mix 1'''<br />
<br />
1. Add RNase free water to reach the final volume of 25 microliter<br><br />
<div style="float: right;"><html><br />
<object style="height: 170px; width: 290px" ><param name="movie" value="http://www.youtube.com/v/vIbKpzlOAXA" ><param name="allowFullScreen" value="true" ><param name="allowScriptAccess" value="always" ><embed src="http://www.youtube.com/v/vIbKpzlOAXA" type="application/x-shockwave-flash" allowfullscreen="true" allowScriptAccess="always" width="290" height="170" ></object><br />
</html></div><br><br><br />
<br />
<br />
2. Add 1 microliter of PCR grade nucleotide mix <br><br><br><br />
<br />
3. Add downstream primer to reach the final concentration of 300 nanomolar<Br><br><br><br />
<br />
<br />
4. Add upstream primer in the same way.<br><br />
<div style="float: right;"><html><br />
<object style="height: 170px; width: 290px" ><param name="movie" value="http://www.youtube.com/v/j-hmoyMSc2Y" ><param name="allowFullScreen" value="true" ><param name="allowScriptAccess" value="always" ><embed src="http://www.youtube.com/v/j-hmoyMSc2Y" type="application/x-shockwave-flash" allowfullscreen="true" allowScriptAccess="always" width="290" height="170" ></object><br />
</html></div><br><Br><br />
<br />
<br />
5. Add Template DNA which contains 10 to 250 nanogram complex DNA <br><br><br><br />
<br />
'''Prepare Master mix 2'''<Br><br><br><br />
<br />
6. Add 19.25 microliter of RNase free water<Br><br />
<div style="float: right;"><html><br />
<object style="height: 170px; width: 290px" ><param name="movie" value="http://www.youtube.com/v/8_cafnNEqhU" ><param name="allowFullScreen" value="true" ><param name="allowScriptAccess" value="always" ><embed src="http://www.youtube.com/v/8_cafnNEqhU" type="application/x-shockwave-flash" allowfullscreen="true" allowScriptAccess="always" width="290" height="170" ></object><br />
</html></div><br><br><br />
<br />
7. Add 5 microliter of Expand High Fidelity Buffer with MgCl2<Br><br><br><br />
<br />
8. Add 0.75 of Expand High Fidelity Enzyme Mix<Br><br><Br><br />
<br />
9. Vortex the components in both Master mixes<Br><br />
<div style="float: right;"><html><br />
<object style="height: 170px; width: 290px" ><param name="movie" value="http://www.youtube.com/v/9fUZtcbOvqs" ><param name="allowFullScreen" value="true" ><param name="allowScriptAccess" value="always" ><embed src="http://www.youtube.com/v/9fUZtcbOvqs" type="application/x-shockwave-flash" allowfullscreen="true" allowScriptAccess="always" width="290" height="170" ></object><br />
</html></div><br><Br><br />
<br />
<br />
10. Prepare the PCR tubes<Br><br><br><br />
<br />
11. Divide the mix1 into the tubes in a desired volume<Br><br><br><br />
<br />
12. Then add the mix2 into the PCR tubes which contain mix 1<Br><br />
<div style="float: right;"><html><br />
<object style="height: 170px; width: 290px" ><param name="movie" value="http://www.youtube.com/v/nDOxB_-aST0" ><param name="allowFullScreen" value="true" ><param name="allowScriptAccess" value="always" ><embed src="http://www.youtube.com/v/nDOxB_-aST0" type="application/x-shockwave-flash" allowfullscreen="true" allowScriptAccess="always" width="290" height="170" ></object><br />
</html></div><br><br><br />
<br />
13. Set up the Thermocycler<br><br><br><br />
<br />
14. Place the PCR tubes into the thermocycler.<br><br />
<br />
<br><br><br><br><br />
<br />
==An example program==<br />
Program a standard thermocycler to run the reaction using the following parameters:<br />
Initial denaturation<br />
• Denature: 96°C, 5 mins<br />
This denatures any double stranded DNA.<br />
Thermocycling<br><br />
• No. of cycles: 25<br><br />
• Denature: 96°C, 1 min<br><br />
• Anneal: 55°C, 30 secs<br><br />
• Elongate: 72°C, 1 min<Br><br />
Termination<br><br />
• Elongate: 72°C, 5 mins<br><br />
• Hold: 4°C, until removed from machine<br><br />
<br />
<br />
<br />
==References==<br />
<br />
1. Arezi B, Xing W, Sorge JA, and Hogrefe HH. Amplification efficiency of thermostable DNA polymerases. Anal Biochem 2003 Oct 15; 321(2) 226-35. pmid:14511688. PubMed HubMed [Arezi-AnalBiochem-2003]<br />
<br />
2. Saiki RK, Scharf S, Faloona F, Mullis KB, Horn GT, Erlich HA, and Arnheim N. Enzymatic amplification of beta-globin genomic sequences and restriction site analysis for diagnosis of sickle cell anemia.Science 1985 Dec 20; 230(4732) 1350-4. pmid:2999980. PubMed HubMed [Saiki-Science-1985]<br />
original paper on PCR<br />
<br />
3. Mullis K, Faloona F, Scharf S, Saiki R, Horn G, and Erlich H. Specific enzymatic amplification of DNA in vitro: the polymerase chain reaction. Cold Spring Harb Symp Quant Biol 1986; 51 Pt 1 263-73.pmid:3472723. PubMed HubMed [Mullis-1986]<br />
first public presentation on PCR</div>Liorhttp://2010.igem.org/Team:Debrecen-Hungary/video_projectTeam:Debrecen-Hungary/video project2010-10-26T20:45:22Z<p>Lior: </p>
<hr />
<div>{{Template:Debrecen-Hungary_secret}}<br />
<br />
<br />
<html><br />
<table width="910" bgcolor="#EDE8E2" border="0" cellspacing="0" cellpadding="0"><br />
<tr><br />
<td align="left" bgcolor="#EDE8E2" border="0" cellspacing="0" cellpadding="0"><br />
<img src="https://static.igem.org/mediawiki/2010/3/33/Video.jpg" width="500" height="150"><br />
</td><br />
<br />
<br />
<td align="right" bgcolor="#EDE8E2" border="0"><br />
<a href="https://2010.igem.org/Team:Debrecen-Hungary"><img src="https://static.igem.org/mediawiki/2010/9/96/Backbuttonbck.jpg" align="right"></a><br />
</td><br />
</tr><br />
</table><br />
<br />
<br />
<br />
<body id="Parts"></body><br />
</html><br />
{|<br />
|-valign="top" border="0" style="margin-left: 2px;"<br />
|width="950px" style="padding: 0 15px 15px 20px; background-color:#ede8e2"|<br />
<br />
<div align="justify"><br />
<br />
="Film In Lab" - The Video Tutorial Project=<br />
<br />
<br />
<br />
<br />
The iGEM – Debrecen group is a multidisciplinary team. In summer 2010 we had guest students, the AKG team, from a high school in Budapest joining us. Our aim was to bring all students, coming from different backgrounds, into a functioning unit. Thus, we have created as part of the preparatory phase the video tutorials. The video tutorials project, also called:"Film in Lab" project, started as an initiative of our group and grew larger.<br />
<br />
<br />
<br />
In general, the idea was to create an observational way of teaching the basic lab techniques. Nevertheless, the project got lots of empathy, thus we have decided to improve it by adding soundtracks, pearls and comments. For this, we have created an own sub team that produced and created the "Film in Lab" project. This way we are aiming to establish an open source that will serve future iGEM members around the globe. We believe that this contribution is in harmony with the idea of the iGEM initiative. <br />
<br />
<br />
<br />
Moreover, this "Film in Lab" project is a great example for how complicated procedures can be simplified; and resulting in efficient application of the theoretical knowledge by a beginner.<br />
For making the experience complete and fruitful, one may find the protocols in the [http://www.openwetware.org/wiki/Special:Contributions/Yakir_Guri 'Open Wet Ware'] account of iGEM-Debrecen Team 2010. We recommend to read the protocols carefully before and after watching the video tutorial for maximizing the experience.<br />
<br />
<br />
<br />
We thank'' 'Roche''' for supporting and sponsoring this project. Also, we thank all students members and supervisors for critical review of scripts and audio soundtracks. Especially, to Dorottya Jakab (AKG team) and Burger Veronika (a sixth year medical student) who made this project posible.<br />
<br />
<br />
See more at our [http://www.youtube.com/user/debrecenigem2010 Youtube channel]<br />
<br />
<br />
<h5>The members of the "Film in Lab" project :</h5><br />
<br />
Dorottya, Jakab – presenting student (Transformation, Midiprep, Gel-Electrophoresis)<br />
<br />
Ozgyin, Lilla – presenting student (Cell-passaging, Ligand stimulation, Transfection)<br />
<br />
Sandor, Kata – presenting student (PCR)<br />
<br />
Nagy, Kata - critical review of Midiprep <br />
<br />
Malka, Lior – presenting student (nanodrop),and our web master<br />
<br />
Endre, Kristof – video making (PCR)<br />
<br />
Guri, Yakir – supervising the project, film making and editing<br />
<br />
Liu, Shun-Chien – coordinator, Scripts, video making (nanodrop) and audio co-editor <br />
<br />
Markovits, Daniel – Audio <br />
<br />
<br><br><br />
<br />
''Guri, Yakir''<Br><br />
<br />
Video Project supervisor <br />
<br />
<br />
<br />
''Balint L. Balint'', MD, PhD,<br />
<br />
Supervisor and instructor of 'iGEM-Debrecen 2010 Team' <br />
<br><br><br><br />
<br />
<html><br />
<center><br />
<img src="https://static.igem.org/mediawiki/2010/b/be/Roche.jpg<br />
"></a><br />
</center><br />
</html><br />
<br />
<br />
<br><br><br><br />
<html><br />
<center><br />
<a href="https://2010.igem.org/Team:Debrecen-Hungary"><img src="https://static.igem.org/mediawiki/2010/9/96/Backbuttonbck.jpg"></a><br />
</center><br />
</html></div>Liorhttp://2010.igem.org/Team:Debrecen-Hungary/protocols/PCRTeam:Debrecen-Hungary/protocols/PCR2010-10-26T20:42:01Z<p>Lior: </p>
<hr />
<div>{{Template:Debrecen-Hungary_secret}}<br />
<br />
<br />
<br />
<br />
==Overview==<br />
<br />
PCR is an acronym for polymerase chain reaction. It is a method for amplifying DNA in vitro.<br />
<br />
==Reagents==<br />
• Expand High Fidelity Buffer with MgCl2<br />
<br />
• PCR grade nucleotide mix MgCl 2<br />
<br />
• Upstream primer<br />
<br />
• Downstream primer<br />
<br />
• Template DNA<br />
<br />
• RNase free water <br />
<br />
• Expand High Fidelity Enzyme Mix<br />
==Equipment==<br />
• Thermocycler<br />
<br />
• Reaction tubes<br />
<br />
==Step==<br />
'''Prepare Master mix 1'''<br />
<br />
1. Add RNase free water to reach the final volume of 25 microliter<br><br><Br><br />
<br />
<br />
2. Add 1 microliter of PCR grade nucleotide mix <br><br />
<div style="float: right;"><html><br />
<object style="height: 170px; width: 290px" ><param name="movie" value="http://www.youtube.com/v/vIbKpzlOAXA" ><param name="allowFullScreen" value="true" ><param name="allowScriptAccess" value="always" ><embed src="http://www.youtube.com/v/vIbKpzlOAXA" type="application/x-shockwave-flash" allowfullscreen="true" allowScriptAccess="always" width="290" height="170" ></object><br />
</html></div><br><br><br />
<br />
3. Add downstream primer to reach the final concentration of 300 nanomolar<Br><br><br><br />
<br />
<br />
4. Add upstream primer in the same way.<br><br><Br><br />
<br />
<br />
5. Add Template DNA which contains 10 to 250 nanogram complex DNA <br><br />
<div style="float: right;"><html><br />
<object style="height: 170px; width: 290px" ><param name="movie" value="http://www.youtube.com/v/j-hmoyMSc2Y" ><param name="allowFullScreen" value="true" ><param name="allowScriptAccess" value="always" ><embed src="http://www.youtube.com/v/j-hmoyMSc2Y" type="application/x-shockwave-flash" allowfullscreen="true" allowScriptAccess="always" width="290" height="170" ></object><br />
</html></div><br><Br><br />
<br />
'''Prepare Master mix 2'''<Br><br><br><br />
<br />
6. Add 19.25 microliter of RNase free water<Br><br><br><br />
<br />
7. Add 5 microliter of Expand High Fidelity Buffer with MgCl2<Br><br />
<div style="float: right;"><html><br />
<object style="height: 170px; width: 290px" ><param name="movie" value="http://www.youtube.com/v/8_cafnNEqhU" ><param name="allowFullScreen" value="true" ><param name="allowScriptAccess" value="always" ><embed src="http://www.youtube.com/v/8_cafnNEqhU" type="application/x-shockwave-flash" allowfullscreen="true" allowScriptAccess="always" width="290" height="170" ></object><br />
</html></div><br><br><br />
<br />
8. Add 0.75 of Expand High Fidelity Enzyme Mix<Br><br><Br><br />
<br />
9. Vortex the components in both Master mixes<Br><Br><br><br />
<br />
10. Prepare the PCR tubes<Br><br />
<div style="float: right;"><html><br />
<object style="height: 170px; width: 290px" ><param name="movie" value="http://www.youtube.com/v/9fUZtcbOvqs" ><param name="allowFullScreen" value="true" ><param name="allowScriptAccess" value="always" ><embed src="http://www.youtube.com/v/9fUZtcbOvqs" type="application/x-shockwave-flash" allowfullscreen="true" allowScriptAccess="always" width="290" height="170" ></object><br />
</html></div><br><Br><br />
<br />
11. Divide the mix1 into the tubes in a desired volume<Br><br><br><br />
<br />
12. Then add the mix2 into the PCR tubes which contain mix 1<Br><br><Br><br />
<br />
13. Set up the Thermocycler<br><br />
<div style="float: right;"><html><br />
<object style="height: 170px; width: 290px" ><param name="movie" value="http://www.youtube.com/v/nDOxB_-aST0" ><param name="allowFullScreen" value="true" ><param name="allowScriptAccess" value="always" ><embed src="http://www.youtube.com/v/nDOxB_-aST0" type="application/x-shockwave-flash" allowfullscreen="true" allowScriptAccess="always" width="290" height="170" ></object><br />
</html></div><br><br><br />
<br />
14. Place the PCR tubes into the thermocycler.<br> <br />
<br><br><br><br><br><br><br />
<br />
==An example program==<br />
Program a standard thermocycler to run the reaction using the following parameters:<br />
Initial denaturation<br />
• Denature: 96°C, 5 mins<br />
This denatures any double stranded DNA.<br />
Thermocycling<br><br />
• No. of cycles: 25<br><br />
• Denature: 96°C, 1 min<br><br />
• Anneal: 55°C, 30 secs<br><br />
• Elongate: 72°C, 1 min<Br><br />
Termination<br><br />
• Elongate: 72°C, 5 mins<br><br />
• Hold: 4°C, until removed from machine<br><br />
<br />
<br />
<br />
==References==<br />
<br />
1. Arezi B, Xing W, Sorge JA, and Hogrefe HH. Amplification efficiency of thermostable DNA polymerases. Anal Biochem 2003 Oct 15; 321(2) 226-35. pmid:14511688. PubMed HubMed [Arezi-AnalBiochem-2003]<br />
<br />
2. Saiki RK, Scharf S, Faloona F, Mullis KB, Horn GT, Erlich HA, and Arnheim N. Enzymatic amplification of beta-globin genomic sequences and restriction site analysis for diagnosis of sickle cell anemia.Science 1985 Dec 20; 230(4732) 1350-4. pmid:2999980. PubMed HubMed [Saiki-Science-1985]<br />
original paper on PCR<br />
<br />
3. Mullis K, Faloona F, Scharf S, Saiki R, Horn G, and Erlich H. Specific enzymatic amplification of DNA in vitro: the polymerase chain reaction. Cold Spring Harb Symp Quant Biol 1986; 51 Pt 1 263-73.pmid:3472723. PubMed HubMed [Mullis-1986]<br />
first public presentation on PCR</div>Liorhttp://2010.igem.org/Team:Debrecen-Hungary/protocols/PCRTeam:Debrecen-Hungary/protocols/PCR2010-10-26T20:39:03Z<p>Lior: </p>
<hr />
<div>{{Template:Debrecen-Hungary_secret}}<br />
<br />
<br />
<br />
<br />
==Overview==<br />
<br />
PCR is an acronym for polymerase chain reaction. It is a method for amplifying DNA in vitro.<br />
<br />
==Reagents==<br />
• Expand High Fidelity Buffer with MgCl2<br />
<br />
• PCR grade nucleotide mix MgCl 2<br />
<br />
• Upstream primer<br />
<br />
• Downstream primer<br />
<br />
• Template DNA<br />
<br />
• RNase free water <br />
<br />
• Expand High Fidelity Enzyme Mix<br />
==Equipment==<br />
• Thermocycler<br />
<br />
• Reaction tubes<br />
<br />
==Step==<br />
'''Prepare Master mix 1'''<br />
<br />
1. Add RNase free water to reach the final volume of 25 microliter<br><br><Br><br />
<br />
<br />
2. Add 1 microliter of PCR grade nucleotide mix <br><br />
<div style="float: right;"><html><br />
<object style="height: 170px; width: 290px" ><param name="movie" value="http://www.youtube.com/v/vIbKpzlOAXA" ><param name="allowFullScreen" value="true" ><param name="allowScriptAccess" value="always" ><embed src="http://www.youtube.com/v/vIbKpzlOAXA" type="application/x-shockwave-flash" allowfullscreen="true" allowScriptAccess="always" width="290" height="170" ></object><br />
</html></div><br><br><br />
<br />
3. Add downstream primer to reach the final concentration of 300 nanomolar<Br><br><br><br />
<br />
<br />
4. Add upstream primer in the same way.<br><br><Br><br />
<br />
<br />
5. Add Template DNA which contains 10 to 250 nanogram complex DNA <br><br />
<div style="float: right;"><html><br />
<object style="height: 170px; width: 290px" ><param name="movie" value="http://www.youtube.com/v/j-hmoyMSc2Y" ><param name="allowFullScreen" value="true" ><param name="allowScriptAccess" value="always" ><embed src="http://www.youtube.com/v/j-hmoyMSc2Y" type="application/x-shockwave-flash" allowfullscreen="true" allowScriptAccess="always" width="290" height="170" ></object><br />
</html></div><br><Br><br />
<br />
'''Prepare Master mix 2'''<Br><br><br><br />
<br />
6. Add 19.25 microliter of RNase free water<Br><br><br><br />
<br />
7. Add 5 microliter of Expand High Fidelity Buffer with MgCl2<Br><br />
<div style="float: right;"><html><br />
<object style="height: 170px; width: 290px" ><param name="movie" value="http://www.youtube.com/v/8_cafnNEqhU" ><param name="allowFullScreen" value="true" ><param name="allowScriptAccess" value="always" ><embed src="http://www.youtube.com/v/8_cafnNEqhU" type="application/x-shockwave-flash" allowfullscreen="true" allowScriptAccess="always" width="290" height="170" ></object><br />
</html></div><br><br><br />
<br />
8. Add 0.75 of Expand High Fidelity Enzyme Mix<Br><br><Br><br />
<br />
9. Vortex the components in both Master mixes<Br><Br><br><br />
<br />
10. Prepare the PCR tubes<Br><br />
<div style="float: right;"><html><br />
<object style="height: 170px; width: 290px" ><param name="movie" value="http://www.youtube.com/v/9fUZtcbOvqs" ><param name="allowFullScreen" value="true" ><param name="allowScriptAccess" value="always" ><embed src="http://www.youtube.com/v/9fUZtcbOvqs" type="application/x-shockwave-flash" allowfullscreen="true" allowScriptAccess="always" width="290" height="170" ></object><br />
</html></div><br><Br><br />
<br />
11. Divide the mix1 into the tubes in a desired volume<Br><br />
<br />
12. Then add the mix2 into the PCR tubes which contain mix 1<Br><br><Br><br />
<br />
13. Set up the Thermocycler<br><br />
<div style="float: right;"><html><br />
<object style="height: 170px; width: 290px" ><param name="movie" value="http://www.youtube.com/v/nDOxB_-aST0" ><param name="allowFullScreen" value="true" ><param name="allowScriptAccess" value="always" ><embed src="http://www.youtube.com/v/nDOxB_-aST0" type="application/x-shockwave-flash" allowfullscreen="true" allowScriptAccess="always" width="290" height="170" ></object><br />
</html></div><br><br><br />
<br />
14. Place the PCR tubes into the thermocycler.<br> <br />
<br><br><br><br><br><br><br />
<br />
==An example program==<br />
Program a standard thermocycler to run the reaction using the following parameters:<br />
Initial denaturation<br />
• Denature: 96°C, 5 mins<br />
This denatures any double stranded DNA.<br />
Thermocycling<br><br />
• No. of cycles: 25<br><br />
• Denature: 96°C, 1 min<br><br />
• Anneal: 55°C, 30 secs<br><br />
• Elongate: 72°C, 1 min<Br><br />
Termination<br><br />
• Elongate: 72°C, 5 mins<br><br />
• Hold: 4°C, until removed from machine<br><br />
<br />
<br />
<br />
==References==<br />
<br />
1. Arezi B, Xing W, Sorge JA, and Hogrefe HH. Amplification efficiency of thermostable DNA polymerases. Anal Biochem 2003 Oct 15; 321(2) 226-35. pmid:14511688. PubMed HubMed [Arezi-AnalBiochem-2003]<br />
<br />
2. Saiki RK, Scharf S, Faloona F, Mullis KB, Horn GT, Erlich HA, and Arnheim N. Enzymatic amplification of beta-globin genomic sequences and restriction site analysis for diagnosis of sickle cell anemia.Science 1985 Dec 20; 230(4732) 1350-4. pmid:2999980. PubMed HubMed [Saiki-Science-1985]<br />
original paper on PCR<br />
<br />
3. Mullis K, Faloona F, Scharf S, Saiki R, Horn G, and Erlich H. Specific enzymatic amplification of DNA in vitro: the polymerase chain reaction. Cold Spring Harb Symp Quant Biol 1986; 51 Pt 1 263-73.pmid:3472723. PubMed HubMed [Mullis-1986]<br />
first public presentation on PCR</div>Liorhttp://2010.igem.org/Team:Debrecen-Hungary/protocols/PCRTeam:Debrecen-Hungary/protocols/PCR2010-10-26T20:24:18Z<p>Lior: </p>
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==Overview==<br />
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PCR is an acronym for polymerase chain reaction. It is a method for amplifying DNA in vitro.<br />
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==Reagents==<br />
• Expand High Fidelity Buffer with MgCl2<br />
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• PCR grade nucleotide mix MgCl 2<br />
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• Upstream primer<br />
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• Downstream primer<br />
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• Template DNA<br />
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• RNase free water <br />
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• Expand High Fidelity Enzyme Mix<br />
==Equipment==<br />
• Thermocycler<br />
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• Reaction tubes<br />
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==Step==<br />
'''Prepare Master mix 1'''<br />
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1. Add RNase free water to reach the final volume of 25 microliter<br><br><Br><br />
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2. Add 1 microliter of PCR grade nucleotide mix <br><br />
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3. Add downstream primer to reach the final concentration of 300 nanomolar<Br><br><br><br />
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4. Add upstream primer in the same way.<br><br><Br><br />
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5. Add Template DNA which contains 10 to 250 nanogram complex DNA <br><br><Br><br />
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'''Prepare Master mix 2'''<Br><br />
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6. Add 19.25 microliter of RNase free water<Br><br />
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7. Add 5 microliter of Expand High Fidelity Buffer with MgCl2<Br><Br><br><br />
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8. Add 0.75 of Expand High Fidelity Enzyme Mix<Br><br><Br><br />
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9. Vortex the components in both Master mixes<Br><br />
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10. Prepare the PCR tubes<Br><br><br> <br />
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11. Divide the mix1 into the tubes in a desired volume<Br><br />
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12. Then add the mix2 into the PCR tubes which contain mix 1<Br><br><Br><br />
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13. Set up the Thermocycler<br><br><br><br />
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14. Place the PCR tubes into the thermocycler.<br> <br />
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==An example program==<br />
Program a standard thermocycler to run the reaction using the following parameters:<br />
Initial denaturation<br />
• Denature: 96°C, 5 mins<br />
This denatures any double stranded DNA.<br />
Thermocycling<br><br />
• No. of cycles: 25<br><br />
• Denature: 96°C, 1 min<br><br />
• Anneal: 55°C, 30 secs<br><br />
• Elongate: 72°C, 1 min<Br><br />
Termination<br><br />
• Elongate: 72°C, 5 mins<br><br />
• Hold: 4°C, until removed from machine<br><br />
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==References==<br />
<br />
1. Arezi B, Xing W, Sorge JA, and Hogrefe HH. Amplification efficiency of thermostable DNA polymerases. Anal Biochem 2003 Oct 15; 321(2) 226-35. pmid:14511688. PubMed HubMed [Arezi-AnalBiochem-2003]<br />
<br />
2. Saiki RK, Scharf S, Faloona F, Mullis KB, Horn GT, Erlich HA, and Arnheim N. Enzymatic amplification of beta-globin genomic sequences and restriction site analysis for diagnosis of sickle cell anemia.Science 1985 Dec 20; 230(4732) 1350-4. pmid:2999980. PubMed HubMed [Saiki-Science-1985]<br />
original paper on PCR<br />
<br />
3. Mullis K, Faloona F, Scharf S, Saiki R, Horn G, and Erlich H. Specific enzymatic amplification of DNA in vitro: the polymerase chain reaction. Cold Spring Harb Symp Quant Biol 1986; 51 Pt 1 263-73.pmid:3472723. PubMed HubMed [Mullis-1986]<br />
first public presentation on PCR</div>Lior