Announcements Quiz Pre lab Lecture Gel Electrophoresis cont Bacterial Transformation Adventures in Troubleshooting Today in Lab M2D3 Announcements Lab notebooks need reasoning and interpretation not just protocol Questions about Quiz 1 Ca2 1 3 primers for SDM 5 reverse complements plasmid Polymerase error rates Taq polymerase 1 in 105 errors in bp Standard version has no proofreading capability exconuclease Pfu polymerase 1 in 106 Standard version requires longer extension times DNA EP Shape dependence Plasmid versus linear samples say 4 Kbp plasmid linear DNA runs with ladder restriction site 2 cut sums to 4 uncut plasmid supercoiled fast circular slow high MW dimers etc Remember to wear nitrile gloves Bacterial transformation keep cold don t vortex 1 chemical treatment competent cells 2 heat shock other methods 1 step electroporation ballistics DNA Amplification in Bacteria Why grow test multiple candidates some plasmids may be incorrect cells that pick up plasmid multicellular colonies Troubleshooting SDM After 2 years of working suddenly no colonies in teaching sample What could be wrong Master Mix problem or changed composition primers 125ng thermal cycler conditions issue template 5 50ng problem with template or inhibitory component SDM control and class data kit control undigested digested primers SDM titration data Red 1 300 IPC gave 50 100 colonies 1 100 IPC sometimes gave a few colonies Blue why would lower template be good competing for other reagents primers etc or inhibitor diluted Today in Lab Set up gel runs 45 min we will photograph it Mark your area with coloured tape Meanwhile notebooks hw etc Finally bacterial transformation be gentle MIT OpenCourseWare http ocw mit edu 20 109 Laboratory Fundamentals in Biological Engineering Spring 2010 For information about citing these materials or our Terms of Use visit http ocw mit edu terms
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