Team:British Columbia/Protocols/Competent Cell Production

From 2012.igem.org

(Difference between revisions)
(Created page with "{{Template:Template_HD_1}}")
 
(11 intermediate revisions not shown)
Line 1: Line 1:
-
{{Template:Template_HD_1}}
+
{{Template:Team:British Columbia Header}}
 +
<html>
 +
<style>
 +
#sponsormap {width:320px;float:left; background-color: white; margin-left: 8px;  margin-top:10px;}
 +
#protocol {width:600px;float:left; background-color: white; margin-left: 8px;  margin-top:10px;}
 +
</style>
 +
<map name="sponsormap">
 +
<area shape="rect" coords="40,40,255,100" href="https://2012.igem.org/Team:British_Columbia/Protocols/Competent_Cell_Production" />
 +
<area shape="rect" coords="40,120,255,180" href="http://partsregistry.org/Help:Protocols/Competent_Cells" />
 +
<area shape="rect" coords="40,200,255,260" href="http://www.neb.com/nebecomm/products/protocol631.asp" />
 +
<area shape="rect" coords="40,280,255,340" href="https://2012.igem.org/Team:British_Columbia/Protocols/Restriction_Digests" />
 +
<area shape="rect" coords="40,360,255,420" href="https://2012.igem.org/Team:British_Columbia/Protocols/Site_Directed_Mutagenesis" />
 +
<area shape="rect" coords="40,440,255,500" href="https://2012.igem.org/Team:British_Columbia/Protocols/GibsonAssembly" />
 +
<area shape="rect" coords="40,520,255,580" href="https://2012.igem.org/Team:British_Columbia/Protocols/DNAPrepBacteria" />
 +
 
 +
<area shape="rect" coords="40,640,255,700" href="https://2012.igem.org/Team:British_Columbia/Protocols/ConsortiaFluor" />
 +
<area shape="rect" coords="40,720,255,780" href="https://2012.igem.org/Team:British_Columbia/Protocols/AArate" />
 +
<area shape="rect" coords="40,800,255,860" href="https://2012.igem.org/Team:British_Columbia/Protocols/Monod" />
 +
<area shape="rect" coords="40,880,255,940" href="https://2012.igem.org/Team:British_Columbia/Protocols/Biodesulfurization" />
 +
</map>
 +
 
 +
<div id="sponsormap"><img align="left" src="https://static.igem.org/mediawiki/2012/d/db/Ubcigemnotebookmenu2.jpg" usemap="#sponsormap" alt="UBC iGEM 2012 protocols"> </div><div id=protocol></html>
 +
<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&deg;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&deg;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&deg;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&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.
 +
#*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.
 +
#'''(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 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.
 +
#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.
 +
#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.
 +
#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.

Latest revision as of 01:37, 4 October 2012

British Columbia - 2012.igem.org
UBC iGEM 2012 protocols

Contents

Competent Cell Production

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.
    • 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.
    • 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.