Team:Debrecen-Hungary/protocols

From 2010.igem.org

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[[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]]
[[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]]
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'''Notebook protocols utilized by the molecular tools subteam '''
'''Notebook protocols utilized by the molecular tools subteam '''
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[[Team:Debrecen-Hungary/protocols#Restriction enzyme digestion|Restriction enzyme digestion]] - [[Team:Debrecen-Hungary/protocols#PCR product purification / Gel purification |PCR Purification]] - [[Team:Debrecen-Hungary/protocols#Gel electrophoresis|Gel electrophoresis]] - [[Team:Debrecen-Hungary/protocols#PCR product purification / Gel purification |Gel purification]]
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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]]  
'''Notebook protocols utilized by the tissue culture subteam'''
'''Notebook protocols utilized by the tissue culture subteam'''
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[[Team:Debrecen-Hungary/protocols#Notebook|[TOP]]]
[[Team:Debrecen-Hungary/protocols#Notebook|[TOP]]]
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=== Cutting gel for PCR product purification ===
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=== Excise agarose gel band for further purification and subcloning ===
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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>
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[http://2010.igem.org/Team:Debrecen-Hungary/protocols/PCR_purification_from_Agarose_Gel Read more...]
[http://2010.igem.org/Team:Debrecen-Hungary/protocols/PCR_purification_from_Agarose_Gel Read more...]
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[[Team:Debrecen-Hungary/protocols#Notebook|[TOP]]]
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=== PCR ===
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<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>
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</html>
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[http://2010.igem.org/Team:Debrecen-Hungary/protocols/PCR Read more...]
[[Team:Debrecen-Hungary/protocols#Notebook|[TOP]]]
[[Team:Debrecen-Hungary/protocols#Notebook|[TOP]]]
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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>
<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>
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[http://2010.igem.org/Team:Debrecen-Hungary/protocols/Gel_electrophoresis Read more...]
[http://2010.igem.org/Team:Debrecen-Hungary/protocols/Gel_electrophoresis Read more...]
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[[Team:Debrecen-Hungary/protocols#Notebook|[TOP]]]
[[Team:Debrecen-Hungary/protocols#Notebook|[TOP]]]
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=== Media PEI Preparation ===
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=== Preparation of DMEM Medium and PEI ===
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. [http://2010.igem.org/Team:Debrecen-Hungary/protocols/_Media_PEI_Preparation Read more...]
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. [http://2010.igem.org/Team:Debrecen-Hungary/protocols/_Media_PEI_Preparation Read more...]
=== Transfection ===
=== Transfection ===
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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.
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[http://2010.igem.org/Team:Debrecen-Hungary/protocols/_Transfection Read more...]
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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>
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</html>
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The following protocol is used to succesfully introduce foreign plasmid DNA into pluripotent cells through a chemical way, fuGENE mediated transfection. [http://2010.igem.org/Team:Debrecen-Hungary/protocols/_Transfection Read more...]
[[Team:Debrecen-Hungary/protocols#Notebook|[TOP]]]
[[Team:Debrecen-Hungary/protocols#Notebook|[TOP]]]
=== Ligand Treatment ===
=== Ligand Treatment ===
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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>
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</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. [http://2010.igem.org/Team:Debrecen-Hungary/protocols/Ligand_Treatment Read more...]
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. [http://2010.igem.org/Team:Debrecen-Hungary/protocols/Ligand_Treatment Read more...]
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[http://www.openwetware.org/wiki/IGEM:University_of_Debrecen:PCR_purification_from_Agarose_Gel ''Main article - PCR product purification / Gel purification''] <br>
[http://www.openwetware.org/wiki/IGEM:University_of_Debrecen:PCR_purification_from_Agarose_Gel ''Main article - PCR product purification / Gel purification''] <br>
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[http://www.openwetware.org/wiki/IGEM:University_of_Debrecen:_Polymerase_Chain_Reaction_%28PCR%29 ''Main article - PCR''] <br>
[http://openwetware.org/wiki/IGEM:University_of_Debrecen:Gel_electrophoresis ''Main article - Gel electrophoresis'']
[http://openwetware.org/wiki/IGEM:University_of_Debrecen:Gel_electrophoresis ''Main article - Gel electrophoresis'']
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[http://openwetware.org/wiki/IGEM:University_of_Debrecen:_Media_PEI_Preparation ''Main article - Media PEI preparation'']
[http://openwetware.org/wiki/IGEM:University_of_Debrecen:_Media_PEI_Preparation ''Main article - Media PEI preparation'']
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[http://www.openwetware.org/wiki/IGEM:University_of_Debrecen:_Transfection ''Main article - Transfection'']
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[http://openwetware.org/wiki/IGEM:University_of_Debrecen:_transfection ''Main article - Transfection'']
[http://openwetware.org/wiki/IGEM:University_of_Debrecen:Ligand_Treatment ''Main article - Ligand treatment'']  
[http://openwetware.org/wiki/IGEM:University_of_Debrecen:Ligand_Treatment ''Main article - Ligand treatment'']  

Latest revision as of 12:20, 27 October 2010



Contents

Welcome To Our Protocols

"Science is nothing else but the art of proper reproducibility. "


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. 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

Contents

Notebook protocols utilized by the bacterial work subteam

Making Lurea's Broth - Transformation of competent cells - Mini Prep - Midi Prep

Notebook protocols utilized by the molecular tools subteam

Restriction enzyme digestion - PCR Purification - PCR - Gel electrophoresis

Notebook protocols utilized by the tissue culture subteam

Cell Passaging - Media PEI Preparation - Transfection - Ligand Treatment

Notebook protocols utilized by the Luciferase subteam

Measuring Luciferase activity with the Victor plate reader

Notebook protocols utilized by the bacterial work subteam

Making Lurea's Broth

Read more...

[TOP]

Transformation of competent cells


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.Read more...

[TOP]

Mini Prep

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 Read more...

Midi Prep

Read more...

[TOP]

Notebook protocols utilized by the molecular tools subteam

Restriction enzyme digestion

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. Read more...

[TOP]

Excise agarose gel band for further purification and subcloning

Read more...

[TOP]

PCR

Read more...

[TOP]

Gel electrophoresis

Read more...

[TOP]

Notebook protocols utilized by the tissue culture subteam

Cell Passaging


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). 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. Read more...

[TOP]

Preparation of DMEM Medium and PEI

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. Read more...

Transfection

The following protocol is used to succesfully introduce foreign plasmid DNA into pluripotent cells through a chemical way, fuGENE mediated transfection. Read more...

[TOP]

Ligand Treatment


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. Read more...

[TOP]

Notebook protocols utilized by the Luciferase subteam

Measuring Luciferase activity with the Victor plate reader

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. Read more...

[TOP]

Online References At Openwetware

Main article - Making Lurea's Broth

Main article - Transformation of competent cells

Main article - Mini prep

Main article - Restriction enzyme digestion

Main article - PCR product purification / Gel purification

Main article - PCR

Main article - Gel electrophoresis

Main article - Cell passaging

Main article - Media PEI preparation

Main article - Transfection

Main article - Ligand treatment

Main article - Measuring Luciferase activity with the Victor plate reader