IGEM:Harvard/2009/Notebook/Harvard iGEM 2010/2010/07/11

iGEM iGarden

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Team Fence Miniprep of Barnase, Gal4 and LacIN ligations No cells found in Barnase colonies 1 and 2 --> innoculations 3 and 4 renamed Barnase 1 and 2, replacing the cell-free cultures. pipetted 4 ml from each overnight cell culture (excepting the cell-free Barnase cultures) into a 15 ml conical *centrifuged at 4400 rpm for 6 minute remaining overnight cell cultures were placed in fridge decanted LB+amp, resuspended cells in 250 μL P1 buffer contents transferred to eppendorfs 250 μL of P2 buffer was added per tube, the tubes were inverted 4-6 times 350 μL N3 buffer added to each, tubes inverted 4-6 times centrifuged for 10 min at 13,000 rpm, supernatant collected ad transferred to QIAprep spin columns centrifuged for 30-60 seconds, flow through discarded 0.5 ml buffer PB added and centrifuged for 30-60 seconds, flow through discarded, spun columns for an additional minute QIAprep columns put into new eppendorfs 50 μL buffer EB added to columns, let stand for 1 minute centrifuged for 1 minute at 13,000 rpm