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Team:British Columbia/Protocols/Competent Cell Production
From 2012.igem.org
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| + | ===Day 1=== | ||
| + | 1. Streak out frozen glycerol stock of bacterial cells (Top 10, DH5α, etc.) onto an LB plate without antibiotics. | ||
| + | |||
| + | 2. Grow plate overnight at 37°C. | ||
| + | |||
| + | ===Day 2=== | ||
| + | 1. Autoclave: | ||
| + | <ul> | ||
| + | <li>2 L of ddH<sub>2</sub>O</li> | ||
| + | <li>100 mL of 10% v/v glycerol (molecular biology grade)</li> | ||
| + | <li>1 L of LB (or preferred media)</li> | ||
| + | <li>4 centrifuge bottles and caps</li> | ||
| + | <li>lots of microfuge tubes</li> | ||
| + | </ul> | ||
| + | |||
| + | 2. Chill overnight at 4°C: | ||
| + | <ul> | ||
| + | <li>ddH<sub>2</sub>O</li> | ||
| + | <li>10% glycerol</li> | ||
| + | <li>centrifuge rotor</li> | ||
| + | </ul> | ||
| + | |||
| + | 3. Prepare starter culture of cells. | ||
| + | <ul> | ||
| + | <li>Select a single colony of E. coli from fresh LB plate and inoculate a 10 mL starter culture of LB (or preferred media).</li> | ||
| + | <li>Grow culture at 37°C in shaker overnight.</li> | ||
| + | </ul> | ||
| + | |||
| + | Notes: | ||
| + | *Possible media substitutes include SOB, 2xYT, etc. | ||
| + | *All glassware should be detergent-free, as trace detergent residue reduces competency. | ||
| + | |||
| + | ===Day 3=== | ||
| + | 1. 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. | ||
| + | |||
| + | 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. | ||
| + | |||
| + | Notes: | ||
| + | *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. | ||
| + | |||
| + | 3. (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. | ||
| + | |||
| + | 4. Decant the supernatant and resuspend each pellet in 200 mL of ice-cold ddH<sub>2</sub>O. | ||
| + | |||
| + | 5. (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. | ||
| + | |||
| + | 6. Decant the supernatant and resuspend each pellet in 100 mL of ice-cold ddH<sub>2</sub>O. | ||
| + | |||
| + | 7. (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. | ||
| + | |||
| + | 8. Decant the supernatant and resuspend each pellet in 40 mL of ice-cold 10% glycerol. Transfer each suspension to a 50 mL conical tube. | ||
| + | |||
| + | 9. 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. | ||
| + | |||
| + | 10. 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. | ||
| + | |||
| + | 11. Aliquot into sterile 1.5 mL microfuge tubes and snap freeze with liquid nitrogen. Store frozen cells in the -80°C freezer. | ||
Revision as of 03:02, 8 June 2012
Day 1
1. Streak out frozen glycerol stock of bacterial cells (Top 10, DH5α, etc.) onto an LB plate without antibiotics.
2. Grow plate overnight at 37°C.
Day 2
1. 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
2. Chill overnight at 4°C:
- ddH2O
- 10% glycerol
- centrifuge rotor
3. 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.
Notes:
- Possible media substitutes include SOB, 2xYT, etc.
- All glassware should be detergent-free, as trace detergent residue reduces competency.
Day 3
1. 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.
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.
Notes:
- 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.
3. (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.
4. Decant the supernatant and resuspend each pellet in 200 mL of ice-cold ddH2O.
5. (SPIN #2) Harvest the cells by centrifugation at 1000g (~2400 rpm in the Beckman JA-10 rotor) for 20 minutes at 4°C.
6. Decant the supernatant and resuspend each pellet in 100 mL of ice-cold ddH2O.
7. (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.
8. Decant the supernatant and resuspend each pellet in 40 mL of ice-cold 10% glycerol. Transfer each suspension to a 50 mL conical tube.
9. 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.
10. 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.
11. Aliquot into sterile 1.5 mL microfuge tubes and snap freeze with liquid nitrogen. Store frozen cells in the -80°C freezer.
