Team:British Columbia/Protocols

E. coli Chemical Transformation

Protocol:

  1. Add 2 uL of plasmid into thawed chemically competent cells.
  2. Incubate for 10-15 minutes on ice.
  3. Heat shock epitube at 42°C for 60 s.
  4. Remove and put on ice for 3 minutes.
  5. Recover with 400 uL of LB media.
  6. Incubate in shaker at 37°C for 1-2 hours.
  7. Plate 150 uL on selective plates, or spin down to increase concentration
  8. Incubate plates at 27°C for 24 hours.

Restriction Enzyme Digestion

Materials:

  • 5 uL buffer
  • 1 ug DNA
  • 1 uL restriction enzyme 1
  • 1 uL restriction enzyme 2
  • up to 50 uL dH20

Protocol:

  1. Incubate mixture at 37°C for 1-2 hours.
  2. Heat inactivate by incubating at 65°C for 15 minutes.

Ligation

Materials:

  • 2 uL T4 ligation buffer(NEB)
  • 2 uL plasmid
  • 8 uL insert
  • 7 uL H20
  • 1 uL ligase

Protocol:

  1. Combine reagents, adding ligase last
  2. Leave mixture at room temperature for 1 hour.
  3. Heat inactivate at 65°C for 10 minutes.

E. coli Chemically Competent Cell Preparation

Materials:

  • 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

Protocol:

  1. Inoculate 100 mL overnight culture in LB at 30°C.
  2. 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.
  3. Chill culture for 10 minutes on ice.
  4. Spin culture for 10-15 minutes at 4000g 4°C.
  5. Remove supernatant and resuspend with 30-40 mL of 0.1 M CaCl2.
  6. Incubate on ice for 30 minutes.
  7. Spin culture down for 10 minutes at 4000g 4°C.
  8. Remove supernatant and resuspend in 6 mL 0.1 M CaCl2 + 15% glycerol.
  9. Aliquot 50 uL in 0.5 mL microfuge tube and snap-freeze in liquid N2.