NanoBio: Prep for Electrocompetent cells: Difference between revisions
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# Pre-chill all tubes, solutions, and cuvettes! | # Pre-chill all tubes, solutions, and cuvettes! | ||
# Pick some colonies from a fresh plate (or back dilute with 20uL from o/n culture) and grow at | # Pick some colonies from a fresh plate (or back dilute with 20uL from o/n culture) and grow at 37°C in 2mL LB+antibiotics. | ||
# If colonies need to be induced, add inducer at OD600 = 0.1 | # If colonies need to be induced, add inducer at OD600 = 0.1 | ||
# Continue growing at 30degC until OD600 = 0.4 - 0.6 | # Continue growing at 30degC until OD600 = 0.4 - 0.6 | ||
Line 26: | Line 26: | ||
=Large-Scale prep= | =Large-Scale prep= | ||
''Rinse all flasks with H<sub>2</sub> | ''Rinse all flasks with H<sub>2</sub>O prior to autoclaving in order to remove residual detergents/salts that may remain on glassware from dishwashing. This step may increase competency. Autoclaving with water, which is then discarded, is even better. '' | ||
# Pre-chill all tubes | # Pre-chill all tubes and solutions! This should include the box and tubes for the cells to be stored in (chill in -80°C) | ||
# Grow an o/n culture of the strain with antibiotics, if appropriate, in LB at 37°C | # Grow an o/n culture of the strain with antibiotics, if appropriate, in LB at 37°C | ||
# Inoculate 500mL fresh LB with the 5mL. Put it back on the shaker at 37°C. | # Inoculate 500mL fresh LB with the 5mL o/n culture. Put it back on the shaker at 37°C. | ||
# Grow until the OD600 is between 0.4 - 0.6. | # Grow until the OD600 is between 0.4 - 0.6. (~3 hours) | ||
# Aliquot 35mL from each sample into 16x 40mL centrifuge tubes. | # Aliquot 35mL from each sample into 16x 40mL centrifuge tubes. | ||
# Chill cells in ice-water bath 15 - 60 min. Increased chill time may increase competency. | # Chill cells in ice-water bath 15 - 60 min. Increased chill time may increase competency. | ||
#*This will subsequently be broken into two batches of 8 tubes. Chill the first batch for 40 minutes. The second batch for 60 minutes. This should give a good rhythm of washing and spinning. | #*This will subsequently be broken into two batches of 8 tubes. Chill the first batch for 40 minutes. The second batch for 60 minutes. This should give a good rhythm of washing and spinning. | ||
# Spin #1: Centrifuge 10m at 4000rcf at 4°C | # Spin #1: Centrifuge 10m at 4000rcf at 4°C | ||
# | # Discard supernatant and resuspend cells in 35mL ice-cold dH<sub>2</sub>O. | ||
# | #*Resuspend gently in 5-10 mL and then add remaining volume. | ||
# Spin #2: Centrifuge 15m at 4000rcf at 4°C | # Spin #2: Centrifuge 15m at 4000rcf at 4°C | ||
# | # Discard supernatant and resuspend cells (GENTLY) in 25mL ice-cold dH<sub>2</sub>O. | ||
#*Resuspend gently in 5-10 mL and then add remaining volume. | |||
# Chill on ice for 30 minutes. | # Chill on ice for 30 minutes. | ||
# Spin #3: Centrifuge 15m at 4000rcf at 4°C | # Spin #3: Centrifuge 15m at 4000rcf at 4°C | ||
# | # Discard supernatant and resuspend cells in 3.5mL ice-cold 10% glycerol. | ||
#*This will not be a good pellet. Be careful when discarding supernatant. (Use pipette. Do not pour off.) | |||
# Chill on ice for 30 minutes. | # Chill on ice for 30 minutes. | ||
# Spin #4: Centrifuge 15m at 4000rcf at 4°C | # Spin #4: Centrifuge 15m at 4000rcf at 4°C | ||
# Remove the supernatant and add 100 μl of 10% glycerol. | # Remove the supernatant and add 100 μl of 10% glycerol. | ||
# Add 10% glycerol to a total volume of ~ | # Add 10% glycerol to a total volume of ~160μL if necessary. (probably won't be!) | ||
# Aliquot 50μL of cell suspensions in pre-chilled tubes. | # Aliquot 50μL of cell suspensions in pre-chilled tubes. | ||
# Shock freeze the tubes in a mixture of dry ice and ethanol. | # Shock freeze the tubes in a mixture of dry ice and ethanol. | ||
Line 52: | Line 54: | ||
# When ready to use, thaw on ice and use immediately. | # When ready to use, thaw on ice and use immediately. | ||
*'''[[User:Heather M. Jensen|Heather M. Jensen]] 18:23, 12 June 2009 (EDT)''': | *'''[[User:Heather M. Jensen|Heather M. Jensen]] 18:23, 12 June 2009 (EDT)''': | ||
Return to [[NanoBio:Protocols|Protocols]]. | Return to [[NanoBio:Protocols|Protocols]]. |
Latest revision as of 11:21, 15 June 2009
There are a couple of tricks to making cells electrocompetent.
- Remove as much salt from the cell suspensions as possible to prevent arcing.
- The cells must remain cold (either 4°C cold room or on ice).
- Once in DI water, the cells become very sensitive to temperature changes and the transformation efficiency drops dramatically if cells are allowed to warm up above 4°C at any step.
Small-scale prep
- Pre-chill all tubes, solutions, and cuvettes!
- Pick some colonies from a fresh plate (or back dilute with 20uL from o/n culture) and grow at 37°C in 2mL LB+antibiotics.
- If colonies need to be induced, add inducer at OD600 = 0.1
- Continue growing at 30degC until OD600 = 0.4 - 0.6
- Aliquot 1mL from each sample into 2x 1.5mL centrifuge tubes
- Chill cells in ice-water bath 10-15min
- Centrifuge 10m at 4000rcf at 4°C
- Note: the centrifuge next to the bioflo cabinet has temp control
- Pipette/discard supernatant and resuspend cells (GENTLY) in 1mL ice-cold dH2O
- Centrifuge 10m at 4000rcf at 4°C
- Resuspend cells (GENTLY) in 0.5mL ice-cold dH2O
- Note: after washing in DI water, the cells become more and more difficult to spin down. Using 10% glycerol can help spin down cells
- Centrifuge 10m at 4000rcf at 4°C
- Resuspend pellet (GENTLY) in 50uL ice-cold water.
Return to Protocols.
Large-Scale prep
Rinse all flasks with H2O prior to autoclaving in order to remove residual detergents/salts that may remain on glassware from dishwashing. This step may increase competency. Autoclaving with water, which is then discarded, is even better.
- Pre-chill all tubes and solutions! This should include the box and tubes for the cells to be stored in (chill in -80°C)
- Grow an o/n culture of the strain with antibiotics, if appropriate, in LB at 37°C
- Inoculate 500mL fresh LB with the 5mL o/n culture. Put it back on the shaker at 37°C.
- Grow until the OD600 is between 0.4 - 0.6. (~3 hours)
- Aliquot 35mL from each sample into 16x 40mL centrifuge tubes.
- Chill cells in ice-water bath 15 - 60 min. Increased chill time may increase competency.
- This will subsequently be broken into two batches of 8 tubes. Chill the first batch for 40 minutes. The second batch for 60 minutes. This should give a good rhythm of washing and spinning.
- Spin #1: Centrifuge 10m at 4000rcf at 4°C
- Discard supernatant and resuspend cells in 35mL ice-cold dH2O.
- Resuspend gently in 5-10 mL and then add remaining volume.
- Spin #2: Centrifuge 15m at 4000rcf at 4°C
- Discard supernatant and resuspend cells (GENTLY) in 25mL ice-cold dH2O.
- Resuspend gently in 5-10 mL and then add remaining volume.
- Chill on ice for 30 minutes.
- Spin #3: Centrifuge 15m at 4000rcf at 4°C
- Discard supernatant and resuspend cells in 3.5mL ice-cold 10% glycerol.
- This will not be a good pellet. Be careful when discarding supernatant. (Use pipette. Do not pour off.)
- Chill on ice for 30 minutes.
- Spin #4: Centrifuge 15m at 4000rcf at 4°C
- Remove the supernatant and add 100 μl of 10% glycerol.
- Add 10% glycerol to a total volume of ~160μL if necessary. (probably won't be!)
- Aliquot 50μL of cell suspensions in pre-chilled tubes.
- Shock freeze the tubes in a mixture of dry ice and ethanol.
- Store at -80°C. These will last between 3-6 months.
- When ready to use, thaw on ice and use immediately.
- Heather M. Jensen 18:23, 12 June 2009 (EDT):
Return to Protocols.