Difference between revisions of "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.

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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.

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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.

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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 N_2.