IGEM:Cambridge/2008/Notebook/Voltage/2008/08/12

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KDP Again try ligation of Kdp and Vector backbone OsmY Again try ligation of OsmY and B0030 backbone This time try using Xba for B0030 and ligate. Gel again and cut out linear fragment. Cut with Spe and gel - cut out circular fragment. Make competent stocks Chemically competent TOP10 cells were made by growing overnight in 200ml LB in a shaking incubater at 37°C. 200ml were spun down at 3800rpm for 8 mins, resuspended in 20ml CaCl2, spun down again and resuspended in 4ml CaCl2. Again they were spun down (6500 rpm for 5 mins)and resuspended in 4ml 60%glycerol, spun down and resuspended in 2ml 60%glycerol. Finally they were aliquoted (100μL) into stock tubes and flash frozen in liquid nitrogen. Electroporation competent TOP10 and DH5α cells were made by growing overnight in 200ml LB in a shaking incubater at 37°C. 100ml were spun down at 3800rpm for 8 mins, resuspended in 25ml SDW, spun down again and resuspended in 2ml SDW. Again they were spun down (6500 rpm for 5 mins)and resuspended in 2ml 60%glycerol, spun down and resuspended in 2ml 60%glycerol. Finally they were aliquoted (100μL) into stock tubes and flash frozen in liquid nitrogen. However lost half of the DH5α when centrifuge tube broke.