E. coli Chemical Transformation
- Add 2 uL of plasmid into thawed chemically competent cells.
- Incubate for 10-15 minutes on ice.
- Heat shock epitube at 42°C for 60 s.
- Remove and put on ice for 3 minutes.
- Recover with 400 uL of LB media.
- Incubate in shaker at 37°C for 1-2 hours.
- Plate 150 uL on selective plates, or spin down to increase concentration
- Incubate plates at 27°C for 24 hours.
Restriction Enzyme Digestion
- 5 uL buffer
- 1 ug DNA
- 1 uL restriction enzyme 1
- 1 uL restriction enzyme 2
- up to 50 uL dH20
- Incubate mixture at 37°C for 1-2 hours.
- Heat inactivate by incubating at 65°C for 15 minutes.
- 2 uL T4 ligation buffer(NEB)
- 2 uL plasmid
- 8 uL insert
- 7 uL H20
- 1 uL ligase
- Combine reagents, adding ligase last
- Leave mixture at room temperature for 1 hour.
- Heat inactivate at 65°C for 10 minutes.
E. coli Chemically Competent Cell Preparation
- LB media
- 0.1 M CaCl2
- 0.1 M CaCl2 + 15% glycerol
- Liquid N2
Keep these on ice for >30 minutes:
- 200 mL centrifuge tubes
- 50 mL Falcon tubes
- 0.5 mL microfuge tubes
- Inoculate 100 mL overnight culture in LB at 30°C.
- Transfer 1 mL overnight culture to 100 mL LB (or scale up) and incubate shaking at 30°C for 3-4 hours until OD600 reaches 0.5-0.6.
- Chill culture for 10 minutes on ice.
- Spin culture for 10-15 minutes at 4000g 4°C.
- Remove supernatant and resuspend with 30-40 mL of 0.1 M CaCl2.
- Incubate on ice for 30 minutes.
- Spin culture down for 10 minutes at 4000g 4°C.
- Remove supernatant and resuspend in 6 mL 0.1 M CaCl2 + 15% glycerol.
- Aliquot 50 uL in 0.5 mL microfuge tube and snap-freeze in liquid N2.