Team:British Columbia/Protocols/Competent Cell Production

From 2012.igem.org

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===Day 1===
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1. Streak out frozen glycerol stock of bacterial cells (Top 10, DH5α, etc.) onto an LB plate without antibiotics.
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2. Grow plate overnight at 37°C.
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 +
===Day 2===
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1. Autoclave:
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<ul>
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<li>2 L of ddH<sub>2</sub>O</li>
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<li>100 mL of 10% v/v glycerol (molecular biology grade)</li>
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<li>1 L of LB (or preferred media)</li>
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<li>4 centrifuge bottles and caps</li>
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<li>lots of microfuge tubes</li>
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</ul>
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2. Chill overnight at 4&deg;C:
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<ul>
 +
<li>ddH<sub>2</sub>O</li>
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<li>10% glycerol</li>
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<li>centrifuge rotor</li>
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</ul>
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3. Prepare starter culture of cells.
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<ul>
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<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>
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<li>Grow culture at 37&deg;C in shaker overnight.</li>
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</ul>
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Notes:
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*Possible media substitutes include SOB, 2xYT, etc.
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*All glassware should be detergent-free, as trace detergent residue reduces competency.
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===Day 3===
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1. Inoculate 1 L of LB media with 10 mL starter culture and grow in a 37&deg;C shaker. Measure the OD<sub>600</sub> every hour, then every 15 - 20 minutes when the OD gets above 0.2.
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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:
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*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&deg;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&deg;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&deg;C.
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 +
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&deg;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&deg;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&deg;C. Start putting 1.5 mL microfuge tubes on ice if not already chilled.
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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.
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11. Aliquot into sterile 1.5 mL microfuge tubes and snap freeze with liquid nitrogen. Store frozen cells in the -80&deg;C freezer.

Revision as of 03:02, 8 June 2012

British Columbia - 2012.igem.org

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.