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Team:Debrecen-Hungary/protocols
From 2010.igem.org
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http://imgur.com/ANxS9.jpg 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 cells[http://www.openwetware.org/wiki/IGEM:University_of_Debrecen:Transformation_of_competent_cells Main 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 Prep[http://openwetware.org/wiki/Editing_IGEM:University_of_Debrecen:_Mini_Prep Main article - Mini prep] Notebook protocols utilized by the molecular tools subteamRestriction enzyme digestion[http://openwetware.org/wiki/IGEM:University_of_Debrecen:Restriction_biobrick_parts#Notes Main 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 purification[http://www.openwetware.org/wiki/IGEM:University_of_Debrecen:PCR_purification_from_Agarose_Gel Main article - PCR product purification / Gel purification] Gel electrophoresis[http://openwetware.org/wiki/IGEM:University_of_Debrecen:Gel_electrophoresis Main article - Gel electrophoresis] Notebook protocols utilized by the tissue culture subteamCell Passaging[http://www.openwetware.org/wiki/IGEM:University_of_Debrecen:Cell_Passaging Main article - Cell passaging]
Media PEI Preparation[http://openwetware.org/wiki/IGEM:University_of_Debrecen:_Media_PEI_Preparation Main 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... Transfection[http://www.openwetware.org/wiki/IGEM:University_of_Debrecen:_Transfection Main article - Transfection] 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. Read more... Ligand Treatment[http://openwetware.org/wiki/IGEM:University_of_Debrecen:Ligand_Treatment Main article - Ligand treatment] Notebook protocols utilized by the Luciferase subteamMeasuring Luciferase activity with the Victor plate reader[http://openwetware.org/wiki/IGEM:University_of_Debrecen:_Enzymatic_activity_with_Victor_machine Main article - easuring Luciferase activity with the Victor plate reader] |
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