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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 - Midi Prep
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 BrothTransformation of competent cellsTransformation 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 PrepThis 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 PrepNotebook protocols utilized by the molecular tools subteamRestriction enzyme digestionBioBrick 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 purificationGel electrophoresisNotebook protocols utilized by the tissue culture subteamCell PassagingCell 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... Media PEI PreparationEukaryotic 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 TreatmentLigand 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... Notebook protocols utilized by the Luciferase subteamMeasuring Luciferase activity with the Victor plate readerhe 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... Online ReferencesMain 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] [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://www.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 - easuring Luciferase activity with the Victor plate reader] |
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