Rutgers:Transformation

From OpenWetWare

Revision as of 22:40, 29 December 2011 by Christopher C Vanlang (Talk | contribs)
(diff) ←Older revision | Current revision (diff) | Newer revision→ (diff)
Jump to: navigation, search

Bacterial transformation(IMPORTANT: keep the cc on ice as much as possible)

  • set a heating block at 42°C and fill wells with DW
  • defrost a 2 ml tube of SOC medium
  • place the required LB plates at 37°C
  • set 15 1.5 ml tubes in a recipient full of watery ice
  • cut the head of a yellow tip with a razor blade to increase its diameter
  • take a tube of XL2-blue competent cells (cc) out of the -80°C freezer, put it immediately in watery ice and wait until it thaws
  • gently stir the cc with the pipette tip, slowly resuspend them
  • distribute 10 µl of cc into 10 of the 15 tubes
  • keep the nulber of tubes needed and put the extra ones at -80°C
  • add 0.18 µl of ß-MAE to each tube
  • incubate on watery ice for 10 min, gently shaking with a finger tip 2-3 times during the 10 min
  • add 1.2 µl of each ligation product into each cc-tubes
  • gently mix
  • incubate on watery ice for 30 min
  • heat shock exactly 30 sec at 42°C in the heating block (no mix, no shake)
  • put on ice during 2 min
  • spin
  • add 125 µl of SOC medium to each tube
  • shake the tubes tilted @ 37°C for 1 h at 225 rpm
  • during this hour, finish preparing the LB plates (stored @ 37°C):

- prepare the appropriate number of Pasteur pipettes close tip and bend - clean bench using EtOH

  • when the incubation is complete, spread 145 µl of SOC-CC onto plate
  • let soak 10 min @ 37°C
  • incubate inverted @ 37°C overnight
  • shift to 4°C for 1 h for color development before screening
  • seal the plates with parafilm and store them at 4°C
Personal tools