Team:Debrecen-Hungary/protocols
From 2010.igem.org
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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.[http://www.openwetware.org/wiki/IGEM:University_of_Debrecen:Transformation_of_competent_cells Read more...] | 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.[http://www.openwetware.org/wiki/IGEM:University_of_Debrecen:Transformation_of_competent_cells Read more...] | ||
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=== Mini Prep === | === Mini Prep === | ||
[http://openwetware.org/wiki/Editing_IGEM:University_of_Debrecen:_Mini_Prep ''Main article - Mini prep''] <br> | [http://openwetware.org/wiki/Editing_IGEM:University_of_Debrecen:_Mini_Prep ''Main article - 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 [http://openwetware.org/wiki/Editing_IGEM:University_of_Debrecen:_Mini_Prep Read more...] | 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 [http://openwetware.org/wiki/Editing_IGEM:University_of_Debrecen:_Mini_Prep Read more...] | ||
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== Notebook protocols utilized by the molecular tools subteam == | == Notebook protocols utilized by the molecular tools subteam == | ||
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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. | 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. | ||
[http://openwetware.org/wiki/IGEM:University_of_Debrecen:Restriction_biobrick_parts#Notes Read more...] | [http://openwetware.org/wiki/IGEM:University_of_Debrecen:Restriction_biobrick_parts#Notes Read more...] | ||
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=== PCR product purification / Gel purification === | === PCR product purification / Gel purification === | ||
[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> | ||
[http://www.openwetware.org/wiki/IGEM:University_of_Debrecen:PCR_purification_from_Agarose_Gel Read more...] | [http://www.openwetware.org/wiki/IGEM:University_of_Debrecen:PCR_purification_from_Agarose_Gel Read more...] | ||
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=== Gel electrophoresis === | === Gel electrophoresis === | ||
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[http://openwetware.org/wiki/IGEM:University_of_Debrecen:Gel_electrophoresis Read more...] | [http://openwetware.org/wiki/IGEM:University_of_Debrecen:Gel_electrophoresis Read more...] | ||
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== Notebook protocols utilized by the tissue culture subteam == | == Notebook protocols utilized by the tissue culture subteam == | ||
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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. | 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. | ||
[http://www.openwetware.org/wiki/IGEM:University_of_Debrecen:Cell_Passaging Read more...] | [http://www.openwetware.org/wiki/IGEM:University_of_Debrecen:Cell_Passaging Read more...] | ||
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=== Media PEI Preparation === | === Media PEI Preparation === |
Revision as of 16:52, 28 September 2010
Welcome To Our Notebook"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 ContentsNotebook protocols utilized by the bacterial work subteam Making Lurea's Broth - Transformation of competent cells - Mini Prep Notebook protocols utilized by the molecular tools subteam Restriction enzyme digestion - PCR Purification - Gel electrophoresis - Gel purification 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 subteamMaking Lurea's BrothMain article - Making Lurea's Broth Transformation of competent cellsMain article - 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... Mini PrepMain article - Mini prep Notebook protocols utilized by the molecular tools subteamRestriction enzyme digestionMain article - 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... PCR product purification / Gel purificationMain article - PCR product purification / Gel purification Gel electrophoresisMain article - Gel electrophoresis Notebook protocols utilized by the tissue culture subteamCell Passaging
Media PEI PreparationMain article - Media PEI preparation 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... TransfectionThe 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. Read more... Ligand TreatmentMain article - Ligand treatment Notebook protocols utilized by the Luciferase subteamMeasuring Luciferase activity with the Victor plate readerMain article - easuring Luciferase activity with the Victor plate reader |
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