Protocol
1 reaction per group
- 25 µL competent cells (get from Tom’s lab, or NEB C2988J)
- 1 µL plasmid Klp61F (10-50 ng) (in plasmids box in 362A freezer) (2024)
- 5 uL annealed plasmid (2025)
- ice 5 minutes
- heat shock 42C 30 sec
- ice, bring volume to 1 mL with LB
- shake (200 rpm) at 37C 20-30 min
- Plate 200 µL on LB-Amp (carbenicillin)
- Make a second plate:
- Spin down remaining 800 µL,
- Resuspend in 200 µL
- 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
- (2025)
- Instructors pick colonies
- (2024)
Students need
- 2 LB-Amp plates per group
- 1 mL LB per group
- sterile water
- sterile beads
- hot block at 42C
- shaker at 37C, rigged with a slanted microfuge tube rack
- (2024) plasmid Klp61F (do miniprep in advance if there is none in the plasmid box in 362A freezer
- (2025) created plasmid
- competent cells
- markers
Day After (instructors)
- Check plates
- Pick colony
- Grow up in liquid culture
- Spin down
- Save pellet for next class