Knight:Preparing electrocompetent cells: Difference between revisions
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*-80°C freezer | *-80°C freezer | ||
*37°C incubator | *37°C incubator | ||
*Refrigerated centrifuge that accepts 225 mL culture tubes | *Refrigerated centrifuge that accepts 225 mL culture tubes | ||
===Chemicals and reagents=== | ===Chemicals and reagents=== | ||
*~500 mL LB Lennox supplemented with appropriate concentration of antibiotic if appropriate. | *~500 mL LB Lennox supplemented with appropriate concentration of antibiotic if appropriate. | ||
*~600 mL sterile deionized water chilled to 4°C | *~600 mL sterile deionized water chilled to 4°C | ||
* | * 50 mL sterile 10% glycerol in deionized water chilled to 4°C | ||
*Ice bucket and ice | *Ice bucket and ice | ||
*Dry ice, ethanol bath | *Dry ice, ethanol bath | ||
===Supplies=== | ===Supplies=== | ||
*Many | *Many 1.5 mL plastic tubes chilled to -80 °C | ||
*14 mL culture tube for starter culture | *14 mL culture tube for starter culture | ||
*2 L flask for culture | *2 L flask for culture | ||
*225 mL plastic tubes for centrifugation | *225 mL plastic tubes for centrifugation | ||
*Pipets | *Pipets | ||
==Procedure== | ==Procedure== | ||
#Prechill all tubes and pipets at 4°C or -80°C as appropriate. <br> Also rinse all flasks with H<sub>2</sub>O prior to autoclaving in order to remove residual detergents that may remain on glassware from dishwashing. This step may increase competency. | #Prechill all tubes and pipets at 4°C or -80°C as appropriate. <br> Also rinse all flasks with H<sub>2</sub>O prior to autoclaving in order to remove residual detergents that may remain on glassware from dishwashing. This step may increase competency. Autoclaving with water, which is then discarded, is even better. | ||
#Inoculate 5mL LB medium and grow overnight at 37°C with rotation. <br> Use LB Lennox rather than LB Miller in order to lessen salt content of media | #Inoculate 5mL LB medium and grow overnight at 37°C with rotation. <br> Use LB Lennox rather than LB Miller in order to lessen salt content of media | ||
# Add the 5mL overnight culture to 450mL LB medium and incubate at 37°C with vigorous shaking until the OD 600nm is between 0.5 and 1.0. It should take about 3 hours. <br> For recA<sup>-</sup> strains, the OD 600 nm should be between 0.5 and 0.7 according to one online source. | # Add the 5mL overnight culture to 450mL LB medium and incubate at 37°C with vigorous shaking until the OD 600nm is between 0.5 and 1.0. It should take about 3 hours. <br> For recA<sup>-</sup> strains, the OD 600 nm should be between 0.5 and 0.7 according to one online source. | ||
# Fast cool the centrifuge with the correct rotor to 4°C | |||
# Pour the culture into two 225 mL centrifuge tubes. | # Pour the culture into two 225 mL centrifuge tubes. | ||
# Place the tubes on ice for 15 minutes. <br> This step can vary in incubation time between 15 minutes and 1 hr. Longer incubation times may lead to higher competency.<br>''For the following steps it is important to keep cells cold and remove all the supernatant in each step to remove residual ions.''<br> | # Place the tubes on ice for 15 minutes. <br> This step can vary in incubation time between 15 minutes and 1 hr. Longer incubation times may lead to higher competency.<br>''For the following steps it is important to keep cells cold and remove all the supernatant in each step to remove residual ions.''<br> | ||
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# Remove supernatant and gently resuspend pellets with 200mL cold sterile water. <br> Initially add 10-20 mL of water and resuspend by pipetting. Then add the rest of the water. | # Remove supernatant and gently resuspend pellets with 200mL cold sterile water. <br> Initially add 10-20 mL of water and resuspend by pipetting. Then add the rest of the water. | ||
# Centrifuge for 15 mins at 2000g at 4°C | # Centrifuge for 15 mins at 2000g at 4°C | ||
# Remove supernatant and gently resuspend pellets with | # Remove supernatant and gently resuspend pellets with 200mL cold sterile water. <br> Initially add 10-20 mL of water and resuspend by pipetting. Then add the rest of the water. | ||
# Hold on ice for 30 minutes | |||
# Centrifuge for 15 mins at 2000g at 4°C | # Centrifuge for 15 mins at 2000g at 4°C | ||
# Remove supernatant and gently resuspend pellets with | # Remove supernatant and gently resuspend pellets with 25mL cold 10% glycerol. <br> This can be optionally transferred to a 50 mL conical tube. | ||
# Hold on ice for 30 minutes | |||
# Centrifuge for 15 mins at 1500g at 4°C | # Centrifuge for 15 mins at 1500g at 4°C | ||
# Remove supernatant and | # Remove the supernatant and add 500 μl of 10% glycerol | ||
# Resuspend the cells in a final volume of approximately 1 ml | |||
# Aliquot 50 μL per tube (tubes on ice) | |||
# Resuspend | |||
# Aliquot 50 μL per tube | |||
# Shock freeze cell suspensions in a dry ice and ethanol bath. <br> One website recommended against using liquid nitrogen but did not justify this recommendation. | # Shock freeze cell suspensions in a dry ice and ethanol bath. <br> One website recommended against using liquid nitrogen but did not justify this recommendation. | ||
# Store at -80°C | # Store at -80°C | ||
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*'''[[User:Rshetty|RS]] 17:49, 13 June 2006 (EDT)''': Dont' forget to use the conical bottoms on the swinging buckets in the centrifuge ... not the flat ones. Otherwise, your tubes will break. (Oops.) | *'''[[User:Rshetty|RS]] 17:49, 13 June 2006 (EDT)''': Dont' forget to use the conical bottoms on the swinging buckets in the centrifuge ... not the flat ones. Otherwise, your tubes will break. (Oops.) | ||
[[Category: | [[Category:Protocol]] | ||
[[Category:In vivo]] | |||
[[Category:Escherichia coli]] | |||
Latest revision as of 13:30, 5 April 2008
Protocol from Austin Che and annotated by Reshma Shetty based on online research.
Materials
Equipment
- -80°C freezer
- 37°C incubator
- Refrigerated centrifuge that accepts 225 mL culture tubes
Chemicals and reagents
- ~500 mL LB Lennox supplemented with appropriate concentration of antibiotic if appropriate.
- ~600 mL sterile deionized water chilled to 4°C
- 50 mL sterile 10% glycerol in deionized water chilled to 4°C
- Ice bucket and ice
- Dry ice, ethanol bath
Supplies
- Many 1.5 mL plastic tubes chilled to -80 °C
- 14 mL culture tube for starter culture
- 2 L flask for culture
- 225 mL plastic tubes for centrifugation
- Pipets
Procedure
- Prechill all tubes and pipets at 4°C or -80°C as appropriate.
Also rinse all flasks with H2O prior to autoclaving in order to remove residual detergents that may remain on glassware from dishwashing. This step may increase competency. Autoclaving with water, which is then discarded, is even better. - Inoculate 5mL LB medium and grow overnight at 37°C with rotation.
Use LB Lennox rather than LB Miller in order to lessen salt content of media - Add the 5mL overnight culture to 450mL LB medium and incubate at 37°C with vigorous shaking until the OD 600nm is between 0.5 and 1.0. It should take about 3 hours.
For recA- strains, the OD 600 nm should be between 0.5 and 0.7 according to one online source. - Fast cool the centrifuge with the correct rotor to 4°C
- Pour the culture into two 225 mL centrifuge tubes.
- Place the tubes on ice for 15 minutes.
This step can vary in incubation time between 15 minutes and 1 hr. Longer incubation times may lead to higher competency.
For the following steps it is important to keep cells cold and remove all the supernatant in each step to remove residual ions. - Centrifuge for 10 mins at 2000g at 4°C
- Remove supernatant and gently resuspend pellets with 200mL cold sterile water.
Initially add 10-20 mL of water and resuspend by pipetting. Then add the rest of the water. - Centrifuge for 15 mins at 2000g at 4°C
- Remove supernatant and gently resuspend pellets with 200mL cold sterile water.
Initially add 10-20 mL of water and resuspend by pipetting. Then add the rest of the water. - Hold on ice for 30 minutes
- Centrifuge for 15 mins at 2000g at 4°C
- Remove supernatant and gently resuspend pellets with 25mL cold 10% glycerol.
This can be optionally transferred to a 50 mL conical tube. - Hold on ice for 30 minutes
- Centrifuge for 15 mins at 1500g at 4°C
- Remove the supernatant and add 500 μl of 10% glycerol
- Resuspend the cells in a final volume of approximately 1 ml
- Aliquot 50 μL per tube (tubes on ice)
- Shock freeze cell suspensions in a dry ice and ethanol bath.
One website recommended against using liquid nitrogen but did not justify this recommendation. - Store at -80°C
Notes
- RS 17:49, 13 June 2006 (EDT): Dont' forget to use the conical bottoms on the swinging buckets in the centrifuge ... not the flat ones. Otherwise, your tubes will break. (Oops.)
