Haynes Lab:Notebook/Engineering PC-TFs/2014/05/22

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Long Transformation
Using the set up ligation products from 5/21.

  • DH5α-T cells were thawed in ice.
  • Three 1.5mL tubes were setup and labeled; 1: CMV/MV9 ligation; 2: CMV/MV9 ligation neg ctrl; 3: RhlI pos ctrl.
  • 50μL of thawed cells were transferred into each of these three empty tubes and pipetted to mix.
  • The two ligation setup products were then transferred into tubes one and two respectively and flicked to mix. Set back on ice.
  • A 1:1000 RhlI plasmid was used as a positive control; 1μL of this plasmid was added to 19μL dH2O to equal the other two products' 20μL total volume. Added to the third tube with 50μL thawed cells.
  • All tubes incubated on ice for 30 minutes.
  • All three tubes were heat shocked on the heat block at 42°C for 30 seconds and then placed back on ice for 2 minutes.
  • 900μL LB broth (no antibiotic) was pipetted into each of the three tubes.
  • Tubes were taped into an empty plate. The plate was taped to the shaker within the incubator.
  • Incubated on shaker for 1 hour at 37°C and 260rpm.
  • Tubes centrifuged for 2 minutes at 6000 x g.
  • 600μL supernatant discarded from each tube.
  • Vortexed tubes to re-suspend cells.
  • 300μL of re-suspended cells were then plates on three marked plates and placed in the incubator for overnight growth.