Transformation (G&GA 2022)

Protocol

1 reaction per group

• 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
  • (2024) For each group, instructors take 3 colonies from transformation plates
  • Grow overnight in LB amp 5 mL aliquots (Thursday)
  • Pellet (Friday), remove supernate. Refrigerate pellet over weekend
  • Distribute pellets Plus miniprep reagents on Monday

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