Team:Debrecen-Hungary/protocols

From 2010.igem.org

(Difference between revisions)
(New page: <html> <body id="Minimals"></body></html> {| |-valign="top" border="0" style="margin-left: 2px;" |width="650px" style="padding: 0 15px 15px 20px; background-color:#ede8e2"| __NOTOC__ <DIV ...)
(→Online References At Openwetware)
 
(126 intermediate revisions not shown)
Line 1: Line 1:
 +
{{Template:Debrecen-Hungary_secret}}
 +
 +
 +
<html>
<html>
-
<body id="Minimals"></body></html>
 
-
{|
 
-
|-valign="top" border="0" style="margin-left: 2px;"
 
-
|width="650px" style="padding: 0 15px 15px 20px; background-color:#ede8e2"|
 
-
__NOTOC__
 
-
<DIV ALIGN=CENTER>
 
-
=Welcome To The Minimals=
+
<table width="910" bgcolor="#EDE8E2" border="0" cellspacing="0" cellpadding="0">
 +
<tr height="180">
 +
<td align="left" bgcolor="#EDE8E2" border="0" cellspacing="0" cellpadding="0" >
 +
<img src="https://static.igem.org/mediawiki/2010/3/3a/Protocols.jpg" width="400" height="110">
 +
</td>
-
From year to year (and from one to jamboree to another) the world of synthetic biology exponentially expands. Some iGEMers may find niches of complex biological systems and use special model organisms or laboratory tools. Our philosophy is that a good project is one that can be kept simple and short. <br>
+
<td align="right" bgcolor="#EDE8E2" border="0">
-
Thus we found it prudent to provide our fellow iGEM teams from abroad with brief overviews of our project background (which we call “the minimals”). Our hope is that it may clear some of the molecular “mish mash” you may be experiencing and help point you in the right direction for a complete understanding of our project.
+
<a href="https://2010.igem.org/Team:Debrecen-Hungary"><img src="https://static.igem.org/mediawiki/2010/9/96/Backbuttonbck.jpg" align="right"></a>
 +
</td>
 +
</tr>
 +
</table>
 +
 +
 +
 +
<body id="Notebook"></body></html>
 +
 +
<div align="justify">
 +
 +
 +
 +
=Welcome To Our Protocols=
 +
 +
<h1> "Science is nothing else but the art of proper reproducibility. " </h1> <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.
 +
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'''==
=='''Contents'''==
-
'''Essentials Of Lipid Sensing'''
+
'''Notebook protocols utilized by the bacterial work subteam '''
-
[[Team:Debrecen-Hungary/minimals#Cellular signaling|Cellular signaling]] - [[Team:Debrecen-Hungary/minimals#Nuclear Receptos|Nuclear Receptors]] -  [[Team:Debrecen-Hungary/minimals#Ligand binding domains|Ligand binding domains]]
+
[[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]]
-
'''Model Organisms'''
+
'''Notebook protocols utilized by the molecular tools subteam '''
-
[[Team:Debrecen-Hungary/minimals#Drosophila Melanogaster|Drosophila Melanogaster]] - [[Team:Debrecen-Hungary/minimals#Caenorhabditis elegans |Caenorhabditis elegans ]] - [[Team:Debrecen-Hungary/minimals#Homo sapiens |Homo sapiens ]]
+
[[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]]  
-
'''In The Laboratory (Techniques And Reagents)'''
+
'''Notebook protocols utilized by the tissue culture subteam'''
-
[[Team:Debrecen-Hungary/minimals#Two Hybrid Screening|Two hybrid screening ]] - [[Team:Debrecen-Hungary/minimals#Luciferase|Luciferase]] - [[Team:Debrecen-Hungary/minimals#Cos-1 cells |Cos-1 cells ]] - [[Team:Debrecen-Hungary/minimals#Dose response curve|Dose response curve ]]
+
[[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 ]]
-
==  Essentials Of Lipid Sensing  ==
+
'''Notebook protocols utilized by the Luciferase subteam'''
-
=== Cellular signaling ===
+
[[Team:Debrecen-Hungary/protocols#Measuring Luciferase activity with the Victor plate reader |Measuring Luciferase activity with the Victor plate reader ]]
-
Cells have an innate ability to “listen” and correctly react to their local or even distant <br> environment. Through time it has been observed that a complex systems of communication governs <br> essential cellular activates and coordinates cell actions.[[Team:Debrecen-Hungary/minimals#References|[1]]] Today, it is well known that processes such as <br>development, growth, tissue repair or death, metabolic shifts and immunity are all governed, at the<br> molecular level, by signaling.  By understanding cell signaling, diseases may be treated effectively and,<br> theoretically, artificial tissues may be created.
+
== Notebook protocols utilized by the bacterial work subteam ==
-
Cells sense information from their local surroundings through a class of proteins known as receptors. Chemicals that activate (or inhibit) receptors are often named hormones, growth factors,  cytokines or even neurotransmitters  yet their proper term is receptor ligands.
+
-
Water soluble ligands have cell membrane penetration and thus mostly interact with trans-membranous receptors, whereas ligands with high lipid solubility easily penetrate the cell membrane
+
-
[[Team:Debrecen-Hungary/minimals#minimals|[TOP]]]
+
=== Making Lurea's Broth===
-
=== Nuclear Receptos ===
+
[https://2010.igem.org/Team:Debrecen-Hungary/protocols/makinglb Read more...]
-
Nuclear receptors are a class of receptors localized within cells which can sense the presence of lipid soluble ligands (eg steroid hormones). Upon ligand binding these receptors undergo a change in conformation and translocate to the nucleus. A unique feature of these proteins, amongst other receptor classes,  is the ability to directly bind to DNA segments known as response elements. These receptors are best viewed as transcription factors which can be activated by extracellular cues[[Team:Debrecen-Hungary/minimals#References|[2]]] [[Team:Debrecen-Hungary/minimals#References|[3]]]. The binding ultimately leads to defined changes in gene expression (both activation and repression), thereby controlling the development, homeostasis, and metabolism of the organism.
+
[[Team:Debrecen-Hungary/protocols#Notebook|[TOP]]]
-
Nuclear receptors bear high homology to each other and are modular into distinct domains: N-terminal regulatory domain, DNA-binding domain, a Hinge region, Ligand binding domain (LBD) and a C-terminal domain.
+
 +
=== Transformation of competent cells ===
 +
<html>
 +
<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>
 +
</html><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...]
-
[[Team:Debrecen-Hungary/minimals#minimals|[TOP]]]
+
[[Team:Debrecen-Hungary/protocols#Notebook|[TOP]]]
-
=== Ligand Binding Domains ===
+
=== 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 [https://2010.igem.org/Team:Debrecen-Hungary/protocols/miniprep Read more...]
-
Ligand binding domain is a conserved sequence and structure amongst the various nuclear receptors whose structure usually referred to as an alpha helical sandwich fold. Three anti parallel alpha helices (the "sandwich filling") are flanked by two alpha helices on one side and three on the other (the "bread"). The ligand cavity has an internal localization just below three anti parallel alpha helical sandwich "filling".  The Ligand binding domain together with the DNA binding domain contributes to the interface of the receptor by binding accessory proteins (coactivator and corepressor) and dimerization of receptors.  
+
-
The LBD also contains the activation function 2 (AF-2) whose action is dependent on the presence of bound ligand [[Team:Debrecen-Hungary/minimals#References|[8]]].
+
-
The change in receptor configuration which occurs upon ligand binding exposes the AF-2 domain, which promotes transcriptional activity by a wide variety of mechanisms.
+
 +
===Midi Prep===
 +
<html>
 +
<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>
 +
</html>
-
[[Team:Debrecen-Hungary/minimals#minimals|[TOP]]]
+
[https://2010.igem.org/Team:Debrecen-Hungary/protocols/midiprep Read more...]
-
== Model Organisms ==
+
[[Team:Debrecen-Hungary/protocols#Notebook|[TOP]]]
-
=== Drosophila Melanogaster ===
+
== Notebook protocols utilized by the molecular tools subteam ==
-
Drosophila Melanogaster, also known as the common fruit fly, is one of the most frequently used model organisms in biological sciences, including studies in genetics, physiology, microbial pathogenesis and life history evolution.[[Team:Debrecen-Hungary/minimals#References|[9]]] 
+
-
The ecdysone receptor is a nuclear receptor found in D.Melanogaster, where it controls development and contributes to other processes such as reproduction. Its ligands are ecdysteroid which are secreted by the organism’s prothoracic gland.
+
 +
=== Restriction enzyme digestion ===
-
[[Team:Debrecen-Hungary/minimals#minimals|[TOP]]]
+
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.
 +
[https://2010.igem.org/Team:Debrecen-Hungary/protocols/Restriction_biobrick_parts Read more...]
-
=== Caenorhabditis elegans===
+
[[Team:Debrecen-Hungary/protocols#Notebook|[TOP]]]
-
Caenorhabditis elegans is a free-living, transparent nematode (roundworm), about 1 mm in length,[[Team:Debrecen-Hungary/minimals#References|[10]]]    which lives in temperate soil environments C. elegans is intensively studied as a model organism in biology for a variety of reasons. The developmental fate of every single somatic cell (959 in the adult hermaphrodite; 1031 in the adult male) has been mapped out.[[Team:Debrecen-Hungary/minimals#References|[11]]] [[Team:Debrecen-Hungary/minimals#References|[12]]]   
+
-
The C.elegans genome harbors 284 nuclear receptors [[Team:Debrecen-Hungary/minimals#References|[10]]]   (a striking figure), which have been shown to control traits such as sex determination, larva development, life span, neuronal growth and identity and much more. As far as nuclear receptors go, they are a gold mine.
+
 +
=== Excise agarose gel band for further purification and subcloning ===
 +
<html>
 +
<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>
 +
</html>
-
[[Team:Debrecen-Hungary/minimals#minimals|[TOP]]]
+
[https://2010.igem.org/Team:Debrecen-Hungary/protocols/PCR_purification_from_Agarose_Gel Read more...]
-
=== Homo Sapiens ===
+
[[Team:Debrecen-Hungary/protocols#Notebook|[TOP]]]
-
Homo sapiens are the only living species in the Homo genus of bipedal primates in  the great ape family. Nuclear receptors number up to 47 in humans, yet only few have been well characterized. They constitute the focus of medicinal reproductive technologies, hormonal medicine (endocrinology), immunology, drug interaction and much more.
+
-
+
-
[[Team:Debrecen-Hungary/minimals#minimals|[TOP]]]
+
-
== In The Laboratory (Techniques And Reagents) ==
+
=== PCR ===
 +
<html>
 +
<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>
 +
</html>
-
=== Two Hybrid Screening ===
+
[https://2010.igem.org/Team:Debrecen-Hungary/protocols/PCR Read more...]
-
Two-hybrid screening is a technique in molecular biology which can be used to investigate protein interaction with other proteins or DNA[[Team:Debrecen-Hungary/minimals#References|[13]]]  [[Team:Debrecen-Hungary/minimals#References|[14]]] by testing for biochemical interactions such as binding.
+
-
The premise behind the test is the activation of reporter gene by a transcription factor binding to DNA response elements located upstream (aka upstream activating sequence or UAS. The transcription factor being investigated is split to two separate functional fragments. The binding domain is the DNA binding domain responsible for associating with the UAS.  The activating domain is responsible for transcriptional activation. When simplified it may be viewed as a biological system at which the input is the transcription factor concentration and the output is the transcriptional activity generated.
+
-
Many versions of the technique have been implemented including one for the study of DNA binding affinity changes in receptors as a cause of ligand binding (the one hybrid screening).
+
-
Yeast Gal 4 is a common DBD used for this techniques purpose. Commly used reporter genes include the product of the LacZ gene (Beta galactosidase) and Luciferase.
+
-
[[Team:Debrecen-Hungary/minimals#minimals|[TOP]]]
+
[[Team:Debrecen-Hungary/protocols#Notebook|[TOP]]]
-
=== Luciferase ===
+
=== Gel electrophoresis ===
-
Luciferase is an enzyme class able to produce bioluminescence by oxidizing the substrate luciferin. "Firefly luciferase" as a laboratory reagent usually refers to P. pyralis luciferase. Its emission can be measured photometrically and hence used to deduce the protein enzyme concentration through standardized methods.
+
<html>
 +
<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>
 +
</html>
-
[[Team:Debrecen-Hungary/minimals#minimals|[TOP]]]
+
[https://2010.igem.org/Team:Debrecen-Hungary/protocols/Gel_electrophoresis Read more...]
-
=== Cos-1 cells  ===
+
[[Team:Debrecen-Hungary/protocols#Notebook|[TOP]]]
-
Cos-1 cells (acronym for CV-1 simian origin, SV-40 viral positive) is cell line derived from the African green monkey kidney cells. It is also often used to transfect cells in tissue culture conditions to produce recombinant proteins for molecular biology, biochemistry, and cell biology experiments. Two forms of COS cell lines commonly used are COS-1 and COS-7. The cell line was obtained by immortalizing the original CV-1 cells with SV-40 virus genome.  This allows the production of large T antigen but has a defect in genomic replication.[[Team:Debrecen-Hungary/minimals#References|[15]]]  
+
-
[[Team:Debrecen-Hungary/minimals#minimals|[TOP]]]
+
== Notebook protocols utilized by the tissue culture subteam ==
-
=== Dose response curve===
+
=== Cell Passaging ===
-
Dose response curve depicts a change in a measured effect on an organism caused by differing levels of exposure to a chemical in standardized measuring conditions. It may apply to either individuals or to populations. The curve is usually displayed in a simple X-Y graph (X being logarithm of dose, Y for effect). The half maximal effective concentration (EC50), a common feature of drug potency, is the chemical’s concentration which induces a response halfway between the baseline and maximum.[[Team:Debrecen-Hungary/minimals#References|[16]]]    
+
<html>
 +
<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>
 +
</html><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).
 +
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.
 +
[https://2010.igem.org/Team:Debrecen-Hungary/protocols/Cell_Passaging Read more...]
-
[[Team:Debrecen-Hungary/minimals#minimals|[TOP]]]
+
[[Team:Debrecen-Hungary/protocols#Notebook|[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. [https://2010.igem.org/Team:Debrecen-Hungary/protocols/_Media_PEI_Preparation Read more...]
 +
=== Transfection ===
 +
<html>
 +
<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>
 +
</html>
-
</DIV>
+
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...]
-
==References==
+
[[Team:Debrecen-Hungary/protocols#Notebook|[TOP]]]
-
<div style="text-align:justify;">
+
 +
=== Ligand Treatment ===
 +
<html>
 +
<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>
 +
</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...]
 +
[[Team:Debrecen-Hungary/protocols#Notebook|[TOP]]]
-
1. ^ Witzany, Guenther (2010). Biocommunication and Natural Genome Editing. Springer. ISBN 9789048133185. <br>
+
== Notebook protocols utilized by the Luciferase subteam ==
-
2. ^ Evans RM (1988). "The steroid and thyroid hormone receptor superfamily". Science 240 (4854): 889–95. doi:10.1126/science.3283939. PMID 3283939.  <br>
+
-
3. ^ Olefsky JM (2001). "Nuclear receptor minireview series". J. Biol. Chem. 276 (40): 36863–4. doi:10.1074/jbc.R100047200. PMID 11459855 begin_of_the_skype_highlighting              11459855      end_of_the_skype_highlighting begin_of_the_skype_highlighting              11459855      end_of_the_skype_highlighting. <br>
+
-
4. ^ a b Mangelsdorf DJ, Thummel C, Beato M, Herrlich P, Schutz G, Umesono K, Blumberg B, Kastner P, Mark M, Chambon P, Evans RM (1995). "The nuclear receptor superfamily: the second decade". Cell 83 (6): 835–9. doi:10.1016/0092-8674(95)90199-X. PMID 8521507. <br>
+
-
5. ^ a b Novac N, Heinzel T (2004). "Nuclear receptors: overview and classification". Curr Drug Targets Inflamm Allergy 3 (4): 335–46. doi:10.2174/1568010042634541. PMID 15584884. http://www.ingentaconnect.com/content/ben/cdtia/2004/00000003/00000004/art00002. <br>
+
-
6. ^ a b c Nuclear Receptors Nomenclature Committee (1999). "A unified nomenclature system for the nuclear receptor superfamily". Cell 97 (2): 161–3. doi:10.1016/S0092-8674(00)80726-6. PMID 10219237. <br>
+
-
7. ^ a b Laudet V (1997). "Evolution of the nuclear receptor superfamily: early diversification from an ancestral orphan receptor". J. Mol. Endocrinol. 19 (3): 207–26. doi:10.1677/jme.0.0190207. PMID 9460643. <br>
+
-
8. ^ a b Wärnmark A, Treuter E, Wright AP, Gustafsson J-Å (2003). "Activation functions 1 and 2 of nuclear receptors: molecular strategies for transcriptional activation". Mol. Endocrinol. 17 (10): 1901–9. doi:10.1210/me.2002-0384. PMID 12893880. <br>
+
-
9. ^ a b Eric C. R. Reeve, ed (2001-06-23). "Drosophila melanogaster: The Fruit Fly". Encyclopedia of genetics. USA: Fitzroy Dearborn Publishers, I. pp. 157. Retrieved 2009-07-01.<br>
+
-
10. WormBook - a free online compendium of all aspects of C. elegans biology, including laboratory protocols
+
-
<br>11. ^ Sulston JE, Horvitz HR (March 1977). "Post-embryonic cell lineages of the nematode, Caenorhabditis elegans". Dev. Biol. 56 (1): 110–56. doi:10.1016/0012-1606(77)90158-0. PMID 838129.
+
-
<br>12. ^ Kimble J, Hirsh D (June 1979). "The postembryonic cell lineages of the hermaphrodite and male gonads in Caenorhabditis elegans". Dev. Biol. 70 (2): 396–417. doi:10.1016/0012-1606(79)90035-6. PMID 478167.
+
-
<br>13. ^ a b c d e f g h i j k l m n o p q r s t u v w x y z aa ab Joung J, Ramm E, Pabo C (2000). "A bacterial two-hybrid selection system for studying protein-DNA and protein-protein interactions". Proc. Natl. Acad. Sci. U.S.A. 97 (13): 7382–7. doi:10.1073/pnas.110149297. PMID 10852947. PMC 16554. http://www.pnas.org/cgi/content/full/97/13/7382.
+
-
<br>14. ^ a b c d e f g h i j Hurt J, Thibodeau S, Hirsh A, Pabo C, Joung J (2003). "Highly specific zinc finger proteins obtained by directed domain shuffling and cell-based selection". Proc. Natl. Acad. Sci. U.S.A. 100 (21): 12271–6. doi:10.1073/pnas.2135381100. PMID 14527993. PMC 218748. http://www.pnas.org/cgi/content/full/100/21/12271.
+
-
<br>15. Gluzman Y (1981) SV40-transformed simian cells support the replication of early SV40 mutants. Cell 23: 175-182. PMID 6260373 <br>
+
-
16. Introducing doseresponse curves, Graphpad Software<br>
+
 +
=== 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. [https://2010.igem.org/Team:Debrecen-Hungary/protocols/_Enzymatic_activity_with_Victor_machine Read more...]
 +
 +
[[Team:Debrecen-Hungary/protocols#Notebook|[TOP]]]
 +
 +
==Online References At Openwetware==
 +
 +
[https://2010.igem.org/Team:Debrecen-Hungary/protocols/makinglb ''Main article - Making Lurea's Broth'']
 +
 +
[http://www.openwetware.org/wiki/IGEM:University_of_Debrecen:Transformation_of_competent_cells ''Main article - Transformation of competent cells'']
 +
 +
[http://openwetware.org/wiki/Editing_IGEM:University_of_Debrecen:_Mini_Prep ''Main article - Mini prep'']
 +
 +
[http://openwetware.org/wiki/IGEM:University_of_Debrecen:Restriction_biobrick_parts#Notes ''Main article - Restriction enzyme digestion'']
 +
 +
[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:_Polymerase_Chain_Reaction_%28PCR%29 ''Main article - PCR''] <br>
 +
 +
[http://openwetware.org/wiki/IGEM:University_of_Debrecen:Gel_electrophoresis ''Main article - Gel electrophoresis'']
 +
 +
[http://www.openwetware.org/wiki/IGEM:University_of_Debrecen:Cell_Passaging ''Main article - Cell passaging'']
 +
 +
[http://openwetware.org/wiki/IGEM:University_of_Debrecen:_Media_PEI_Preparation ''Main article - Media PEI preparation'']
 +
 +
[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:_Enzymatic_activity_with_Victor_machine ''Main article - Measuring Luciferase activity with the Victor plate reader'']
</div>
</div>
-
|width="250px" style="padding: 0 20px 15px 15px; background-color:#d8d5d0"|
 
-
|} </html>
+
<html>
 +
<center>
 +
<a href="https://2010.igem.org/Team:Debrecen-Hungary"><img src="https://static.igem.org/mediawiki/2010/9/96/Backbuttonbck.jpg"></a>
 +
</center>
 +
</html>

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


Retrieved from "http://2010.igem.org/Team:Debrecen-Hungary/protocols"