Team:British Columbia/Protocols/Competent Cell Production

From 2012.igem.org

(Difference between revisions)
m (Reformatted to Wiki-markup)
Line 2: Line 2:
===Day 1===
===Day 1===
-
1. Streak out frozen glycerol stock of bacterial cells (Top 10, DH5α, etc.) onto an LB plate without antibiotics.
+
#Streak out frozen glycerol stock of bacterial cells (Top 10, DH5α, etc.) onto an LB plate without antibiotics.
-
 
+
#Grow plate overnight at 37°C.
-
2. Grow plate overnight at 37°C.
+
===Day 2===
===Day 2===
-
1. Autoclave:
+
#Autoclave:
-
<ul>
+
#*2 L of ddH<sub>2</sub>O
-
<li>2 L of ddH<sub>2</sub>O</li>
+
#*100 mL of 10% v/v glycerol (molecular biology grade)
-
<li>100 mL of 10% v/v glycerol (molecular biology grade)</li>
+
#*1 L of LB (or preferred media)
-
<li>1 L of LB (or preferred media)</li>
+
#*4 centrifuge bottles and caps
-
<li>4 centrifuge bottles and caps</li>
+
#*lots of microfuge tubes
-
<li>lots of microfuge tubes</li>
+
#Chill overnight at 4&deg;C:
-
</ul>
+
#*ddH<sub>2</sub>O
-
 
+
#*10% glycerol
-
2. Chill overnight at 4&deg;C:
+
#*centrifuge rotor
-
<ul>
+
#Prepare starter culture of cells.  
-
<li>ddH<sub>2</sub>O</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>10% glycerol</li>
+
#*Grow culture at 37&deg;C in shaker overnight.
-
<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&deg;C in shaker overnight.</li>
+
-
</ul>
+
-
 
+
Notes:  
Notes:  
-
*Possible media substitutes include SOB, 2xYT, etc.
+
#**Possible media substitutes include SOB, 2xYT, etc.
-
*All glassware should be detergent-free, as trace detergent residue reduces competency.
+
#**All glassware should be detergent-free, as trace detergent residue reduces competency.
===Day 3===
===Day 3===
-
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.
+
#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.
-
 
+
#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.
-
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.
-
Notes:
+
#**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.
-
*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.
+
#'(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.
-
*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.
+
#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&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.
+
#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&deg;C. At this step, rinse two 50 mL conical tubes with ddH<sub>2</sub>O and chill on ice.
-
4. Decant the supernatant and resuspend each pellet in 200 mL of ice-cold ddH<sub>2</sub>O.
+
#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&deg;C. Start putting 1.5 mL microfuge tubes on ice if not already chilled.
-
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.
+
#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&deg;C freezer.
-
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.
+
-
 
+
-
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&deg;C freezer.
+

Revision as of 03:53, 15 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.