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

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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