Difference between revisions of "Team:William and Mary/Protocols"
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<div class="col-sm-8" style = 'padding-right: 30px; padding-left: 205px; line-height: 25px;' >All time course measurements were carried out in the <i>E. coli</i> strain NEB 10-Beta (ΔAra-Leu) in M9 media with .1% casamino acids, .4% glucose (or glycerol when arabinose was used), and 500mM L-leucine, and were measured using a BioRad S3e Cell Sorter, with a minimum of 10,000 cells measured from each of three biological replicates (colonies) collected on the same day. Care was made to maintain cells in midlog growth phase. </div> | <div class="col-sm-8" style = 'padding-right: 30px; padding-left: 205px; line-height: 25px;' >All time course measurements were carried out in the <i>E. coli</i> strain NEB 10-Beta (ΔAra-Leu) in M9 media with .1% casamino acids, .4% glucose (or glycerol when arabinose was used), and 500mM L-leucine, and were measured using a BioRad S3e Cell Sorter, with a minimum of 10,000 cells measured from each of three biological replicates (colonies) collected on the same day. Care was made to maintain cells in midlog growth phase. </div> | ||
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We diluted 100µL of cell culture into 400 µL of Phosphate Buffered Saline (PBS). For our induction experiments, we split and induced the cells with IPTG 6 hours after they were inoculated. Four hours after the cells were induced, we diluted the cells in order to obtain an ideal concentration of OD600 0.01. The cells were then grown for another five hours and taken out every 20 minutes to be filtered on ice and FACSed immediately. To acquire data with flow cytometry, we adjust side-scatter (SSC) and | We diluted 100µL of cell culture into 400 µL of Phosphate Buffered Saline (PBS). For our induction experiments, we split and induced the cells with IPTG 6 hours after they were inoculated. Four hours after the cells were induced, we diluted the cells in order to obtain an ideal concentration of OD600 0.01. The cells were then grown for another five hours and taken out every 20 minutes to be filtered on ice and FACSed immediately. To acquire data with flow cytometry, we adjust side-scatter (SSC) and | ||
forward scatter (FSC) PMT voltages using bacteria from one of our samples until the distribution of each | forward scatter (FSC) PMT voltages using bacteria from one of our samples until the distribution of each | ||
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have been calibrated for excitation and detection of the particles in most channels of any flow | have been calibrated for excitation and detection of the particles in most channels of any flow | ||
cytometer. Every 100 minutes, we diluted the cells 1:10 in pre-warmed culture. We used FlowCal for downstream analysis and conversion to absolute fluorescence units. The two | cytometer. Every 100 minutes, we diluted the cells 1:10 in pre-warmed culture. We used FlowCal for downstream analysis and conversion to absolute fluorescence units. The two | ||
| − | absolute fluorescence units we used are Molecules of Equivalent Fluorescein (MEFL) for GFP and MEPTR for mScarlet.</div> | + | absolute fluorescence units we used are Molecules of Equivalent Fluorescein (MEFL) for GFP and MEPTR for mScarlet.</div |
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| + | <b><div = 'padding-right: 10px;padding-left: 20px; line-height: 25px;' > Additional protocols: </div class></b> | ||
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| + | <div class="col-sm-4" style = 'padding-right: 10px;padding-left: 20px; line-height: 25px;' > <a href="https://static.igem.org/mediawiki/2017/3/31/T--William_and_Mary--PCR.pdf"> PCR </a> </div class> | ||
| + | <div class="col-sm-4" style = 'padding-right: 10px;padding-left: 20px; line-height: 25px;' > <a href="https://static.igem.org/mediawiki/2017/6/60/T--William_and_Mary--GelElectrophoresis1.pdf"> Gel Electrophoresis </a> </div class> | ||
| + | <div class="col-sm-4" style = 'padding-right: 10px;padding-left: 20px; line-height: 25px;' > <a href="https://static.igem.org/mediawiki/2017/9/94/T--William_and_Mary--DPNI.pdf"> DpnI Digestion </a> </div class> | ||
| + | <div class="col-sm-4" style = 'padding-right: 10px;padding-left: 20px; line-height: 25px;' > <a href="https://static.igem.org/mediawiki/2017/0/03/T--William_and_Mary--PCRpurification.pdf"> PCR Purification</a> </div class> | ||
| + | <div class="col-sm-4" style = 'padding-right: 10px;padding-left: 20px; line-height: 25px;' > <a href="https://static.igem.org/mediawiki/2017/d/d5/T--William_and_Mary--Gibson_Assembly.pdf"> Gibson Assembly </a> </div class> | ||
| + | <div class="col-sm-4" style = 'padding-right: 10px;padding-left: 20px; line-height: 25px;' > <a href="https://static.igem.org/mediawiki/2017/9/92/T--William_and_Mary--Transformation.pdf">Chemical Transformation </a> </div class> | ||
| + | <div class="col-sm-4" style = 'padding-right: 10px;padding-left: 20px; line-height: 25px;' > <a href="https://static.igem.org/mediawiki/2017/4/41/T--William_and_Mary--Inoculation.pdf"> Inoculation </a> </div class> | ||
| + | <div class="col-sm-4" style = 'padding-right: 10px;padding-left: 20px; line-height: 25px;' > <a href="https://static.igem.org/mediawiki/2017/4/4e/T--William_and_Mary--QiagenMiniprep.pdf"> Qiagen Miniprep</a> </div class> | ||
| + | <div class="col-sm-4" style = 'padding-right: 10px;padding-left: 20px; line-height: 25px;' > <a href="https://static.igem.org/mediawiki/2017/3/39/T--William_and_Mary--Plate_Reader.pdf"> Plate Reader </a> </div class> | ||
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</div> | </div> | ||
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<div style='padding-top: 100px;'></div> | <div style='padding-top: 100px;'></div> | ||
Revision as of 18:13, 1 November 2017
