Team:British Columbia/Protocols/Competent Cell Production
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+ | <h1>Competent Cell Production</h1> | ||
+ | |||
+ | <h2>Day 1</h2> | ||
+ | #Streak out frozen glycerol stock of bacterial cells (Top 10, DH5α, etc.) onto an LB plate without antibiotics. | ||
+ | #Grow plate overnight at 37°C. | ||
+ | |||
+ | <h2>Day 2</h2> | ||
+ | #Autoclave: | ||
+ | #*2 L of ddH<sub>2</sub>O | ||
+ | #*100 mL of 10% v/v glycerol (molecular biology grade) | ||
+ | #*1 L of LB (or preferred media) | ||
+ | #*4 centrifuge bottles and caps | ||
+ | #*lots of microfuge tubes | ||
+ | #Chill overnight at 4°C: | ||
+ | #*ddH<sub>2</sub>O | ||
+ | #*10% glycerol | ||
+ | #*centrifuge rotor | ||
+ | #Prepare starter culture of cells. | ||
+ | #*Select a single colony of E. coli from fresh LB plate and inoculate a 10 mL starter culture of LB (or preferred media). | ||
+ | #*Grow culture at 37°C in shaker overnight. | ||
+ | #*Possible media substitutes include SOB, 2xYT, etc. | ||
+ | #*All glassware should be detergent-free, as trace detergent residue reduces competency. | ||
+ | |||
+ | <h2>Day 3</h2> | ||
+ | #Inoculate 1 L of LB media with 10 mL starter culture and grow in a 37°C shaker. Measure the OD<sub>600</sub> every hour, then every 15 - 20 minutes when the OD gets above 0.2. | ||
+ | #When the OD<sub>600</sub> reaches 0.35 - 0.4, immediately put the cells on ice. Chill the culture for 20 - 30 minutes, swirling occasionally to ensure even cooling. Place centrifuge bottles on ice at this time. | ||
+ | #*It is important not to let the OD get any higher than 0.4. It usually takes about 3 hours to reach an OD of 0.35 when using a 10 mL starter culture. | ||
+ | #*It is also very important to keep the cells at 4°C for the remainder of the procedure. The cells, and any bottles or solutions that they come into contact with, must be pre-chilled to 4°C. | ||
+ | #'''(SPIN #1)''' Split the 1 L culture into four parts by pouring about 250 mL into ice-cold centrifuge bottles. Harvest the cells by centrifugation at 1000<i>g</i> (~2400 rpm in the Beckman JA-10 rotor) for 20 minutes at 4°C. | ||
+ | #Decant the supernatant and resuspend each pellet in 200 mL of ice-cold ddH<sub>2</sub>O. | ||
+ | #'''(SPIN #2)''' Harvest the cells by centrifugation at 1000<i>g</i> (~2400 rpm in the Beckman JA-10 rotor) for 20 minutes at 4°C. | ||
+ | #Decant the supernatant and resuspend each pellet in 100 mL of ice-cold ddH<sub>2</sub>O. | ||
+ | #'''(SPIN #3)''' Combine resuspensions into 2 centrifuge bottles (so each contains about 200 mL of cell suspension). Harvest the cells by centrifugation at 1000<i>g</i> (~2400 rpm in the Beckman JA-10 rotor) for 20 minutes at 4°C. At this step, rinse two 50 mL conical tubes with ddH<sub>2</sub>O and chill on ice. | ||
+ | #Decant the supernatant and resuspend each pellet in 40 mL of ice-cold 10% glycerol. Transfer each suspension to a 50 mL conical tube. | ||
+ | #Harvest the cells by centrifugation at 1000<i>g</i> (~2100 rpm in the Beckman GH-3.8 rotor) for 20 minutes at 4°C. Start putting 1.5 mL microfuge tubes on ice if not already chilled. | ||
+ | #Carefully aspirate the supernatant with a sterile Pasteur pipette (pellets lose adherence in 10% glycerol). Resuspend each pellet in 1 mL of ice-cold 10% glycerol by gently swirling. The final OD<sub>600</sub> of the resuspended cells should be ~ 200 - 250. | ||
+ | #Aliquot into sterile 1.5 mL microfuge tubes and snap freeze with liquid nitrogen. Store frozen cells in the -80°C freezer. |
Latest revision as of 01:37, 4 October 2012
Contents |
Competent Cell Production
Day 1
- Streak out frozen glycerol stock of bacterial cells (Top 10, DH5α, etc.) onto an LB plate without antibiotics.
- Grow plate overnight at 37°C.
Day 2
- Autoclave:
- 2 L of ddH2O
- 100 mL of 10% v/v glycerol (molecular biology grade)
- 1 L of LB (or preferred media)
- 4 centrifuge bottles and caps
- lots of microfuge tubes
- Chill overnight at 4°C:
- ddH2O
- 10% glycerol
- centrifuge rotor
- Prepare starter culture of cells.
- Select a single colony of E. coli from fresh LB plate and inoculate a 10 mL starter culture of LB (or preferred media).
- Grow culture at 37°C in shaker overnight.
- Possible media substitutes include SOB, 2xYT, etc.
- All glassware should be detergent-free, as trace detergent residue reduces competency.
Day 3
- Inoculate 1 L of LB media with 10 mL starter culture and grow in a 37°C shaker. Measure the OD600 every hour, then every 15 - 20 minutes when the OD gets above 0.2.
- When the OD600 reaches 0.35 - 0.4, immediately put the cells on ice. Chill the culture for 20 - 30 minutes, swirling occasionally to ensure even cooling. Place centrifuge bottles on ice at this time.
- It is important not to let the OD get any higher than 0.4. It usually takes about 3 hours to reach an OD of 0.35 when using a 10 mL starter culture.
- It is also very important to keep the cells at 4°C for the remainder of the procedure. The cells, and any bottles or solutions that they come into contact with, must be pre-chilled to 4°C.
- (SPIN #1) Split the 1 L culture into four parts by pouring about 250 mL into ice-cold centrifuge bottles. Harvest the cells by centrifugation at 1000g (~2400 rpm in the Beckman JA-10 rotor) for 20 minutes at 4°C.
- Decant the supernatant and resuspend each pellet in 200 mL of ice-cold ddH2O.
- (SPIN #2) Harvest the cells by centrifugation at 1000g (~2400 rpm in the Beckman JA-10 rotor) for 20 minutes at 4°C.
- Decant the supernatant and resuspend each pellet in 100 mL of ice-cold ddH2O.
- (SPIN #3) Combine resuspensions into 2 centrifuge bottles (so each contains about 200 mL of cell suspension). Harvest the cells by centrifugation at 1000g (~2400 rpm in the Beckman JA-10 rotor) for 20 minutes at 4°C. At this step, rinse two 50 mL conical tubes with ddH2O and chill on ice.
- Decant the supernatant and resuspend each pellet in 40 mL of ice-cold 10% glycerol. Transfer each suspension to a 50 mL conical tube.
- Harvest the cells by centrifugation at 1000g (~2100 rpm in the Beckman GH-3.8 rotor) for 20 minutes at 4°C. Start putting 1.5 mL microfuge tubes on ice if not already chilled.
- Carefully aspirate the supernatant with a sterile Pasteur pipette (pellets lose adherence in 10% glycerol). Resuspend each pellet in 1 mL of ice-cold 10% glycerol by gently swirling. The final OD600 of the resuspended cells should be ~ 200 - 250.
- Aliquot into sterile 1.5 mL microfuge tubes and snap freeze with liquid nitrogen. Store frozen cells in the -80°C freezer.