IGEM:Cambridge/2008/Notebook/Bacillus/2008/07/28

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Results from previous days Results of antibiotic plates from yesterday For Cm, 35μg/mL should be enough to kill B.S. For Amp, nothing can be concluded! Wet Work Check plasmid ppL82 We had 2 samples of ppL82 in DH5α in LB solution (~4mL), one from the frozen glycerol tube and one from a colony picked on a plate. Normally plasmid and cells should not be kept in freezer. So, we want to extract this plasmid, check its size and then keep it in the fridge. We do that for the 2 different samples. - Plasmid Miniprep (standard protocol) - Test the concentration of DNA in each tube ppL82 plate : 89.8ng/mL ppL82 tube : 71ng/mL Preparation of different stocks of strains and plasmids PNZ8901 - Plate a single colony (from 23/07/08) on a Cm plate - Incubate at 37°C - Grow the entire frozen glycerol tube in 20mL of LB without antibiotic to check if this stock is still good (we will check the plasmid on a gel tomorrow) - Incubate at 37°C MC1061 -Pick e single colony of MC1061 (from 22/07/08) and put it in 10mL of LB (to make glycerol stocks) -Incubate at 37°C 1A1 - Add 100μL of LB+1A (mix of friday) and 10mL of LB - Incubate at 37°C Make B.S. 1A1 competent and transform them - Prepare Medium A Add 81mL of SDW, 10mL of 10X Medium A base and 9mL of 10X Bacillus salts - Make B.S. 1A1 competent In 10mL of medium A and add about 10 colonies of B.S. Check the OD650, you should have an OD between 0.1 and 0.2. t0 for OD = 0.1876 Check the OD650 every 20min and plot OD against time on semi-log paper. When the point at the culture leaves log growth, it is ok Time (min) 0 20 40 60 70 80 OD650 0.1876 0.2074 0.3282 0.4545 0.4895 0.5040 Log of OD650 versus time for Competent Bacillus Cell Culture At, t0 = 70min, log growth seems to stop. Incubate at 37°C 90min after t0 Warn 10 Ependorf tubes with 0.45mL of Medium B Add 50μL of culture in each tube of Medium B at t90 Incubate the diluted culture at 37°C with vigorous aeration for 90min - Storage : we want to store 7 tubes 6 tubes in the freezer (with 60μL of glycerol) 1 tube on the bench - Transformation We make ppL82 from plate, ppL82 from tube, and a control Add 0.5μg of DNA (15μL) Incubate at 37°C for 30min Plate on Cm plates