Transformation (G&GA 2022)

Submitted by kdorfman on Thu, 09/15/2022 - 15:11

Protocol

  • 25 uL competent cells l^*] (get from Tom's lab, or NEB C2988J)
  • 1 uL plasmid Klp61F (10-50 ng) (in plasmids box in 362A freezer)
  • ice 5 minutes
  • heat shock 42C 30sec
  • ice, bring volume to 1 mL with LB
  • shake (200 rpm) at 37C 20-30 min
  • Plate 200 uL on LB-Amp
  • Make a second plate:
    • Spin down remaining 800 uL,
    • Resuspend in 200 uL
    • plate on LB-Amp
  • incubate 37C overnight
  • instructors take plates and start liquid cultures from them for the students

Students need

  • 2 LB-Amp plates per pair

  • 1 mL LB per pair

  • sterile water

  • sterile beads

  • hot block at 42C

  • shaker at 37C, rigged with a slanted microfuge tube rack

  • plasmid Klp61F (do miniprep in advance if there is none in the plasmid box in 362A freezer

  • competent cells

  • markers