15 Cloning Vectors
239
Materials
• One colony of an E. coli strain (e.g. LE392, MC1061, HB101)
Reagents
• Recombinant plasmid in a low-salt buffer
• LB agar plates containing the appropriate antibiotics
• LB medium (see Subprotocol 1, Materials)
Solutions
• 10% glycerol solution, sterile-filtered (ice-cold)
• SOC medium (2% Bacto-tryptone, 0.5% Bacto-yeast extract, 10 mM
NaCl, 2.5 mM KCl, 10 mM MgCh, 10 mM MgS04, 20 mM glucose)
• Shaker
Supplies
• Centrifuge
• Spectrophotometer
• Gene Pulser system (Bio-Rad Laboratories)
• 0.2 cm electroporation cuvette (Bio-Rad Laboratories)
• sterile toothpicks
Procedure
1. Prepare an overnight culture of the chosen E. coli strain: To a sterile cul- ln advance
ture flask add 10 ml of LB medium, and inoculate the medium with a
single colony of the E. coli strain. Incubate the culture flask in a shaker
at 37°C overnight.
2. Add 990 ml of LB medium to a sterile culture flask and inoculate the Cell preparation
medium with 10 ml of the overnight culture from step 1. Incubate the
culture at 37°C shaking vigorously.
3. Grow the cells to an OD600 of 0.5-0.7 (log-phase). This requires approximately 3 hrs.
4. Place the cells briefly on ice.
5. Centrifuge the cells at 4000 x g for 15 min at 4°C.
6. Discard the supernatant and resuspend the cells in 300 ml of ice-cold 10%
glycerol solution.
239
Materials
• One colony of an E. coli strain (e.g. LE392, MC1061, HB101)
Reagents
• Recombinant plasmid in a low-salt buffer
• LB agar plates containing the appropriate antibiotics
• LB medium (see Subprotocol 1, Materials)
Solutions
• 10% glycerol solution, sterile-filtered (ice-cold)
• SOC medium (2% Bacto-tryptone, 0.5% Bacto-yeast extract, 10 mM
NaCl, 2.5 mM KCl, 10 mM MgCh, 10 mM MgS04, 20 mM glucose)
• Shaker
Supplies
• Centrifuge
• Spectrophotometer
• Gene Pulser system (Bio-Rad Laboratories)
• 0.2 cm electroporation cuvette (Bio-Rad Laboratories)
• sterile toothpicks
Procedure
1. Prepare an overnight culture of the chosen E. coli strain: To a sterile cul- ln advance
ture flask add 10 ml of LB medium, and inoculate the medium with a
single colony of the E. coli strain. Incubate the culture flask in a shaker
at 37°C overnight.
2. Add 990 ml of LB medium to a sterile culture flask and inoculate the Cell preparation
medium with 10 ml of the overnight culture from step 1. Incubate the
culture at 37°C shaking vigorously.
3. Grow the cells to an OD600 of 0.5-0.7 (log-phase). This requires approximately 3 hrs.
4. Place the cells briefly on ice.
5. Centrifuge the cells at 4000 x g for 15 min at 4°C.
6. Discard the supernatant and resuspend the cells in 300 ml of ice-cold 10%
glycerol solution.