Preparing chemically competent cells (Inoue): Difference between revisions
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==Glassware & equipment== | ==Glassware & equipment== | ||
* 2 liter erlenmeyer flask | * 2 liter erlenmeyer flask (no detergent residue, rinse with 70% ethanol and DI water) | ||
* 220 ml conical centrifuge tubes BD 35 2075 | * 220 ml conical centrifuge tubes BD 35 2075 | ||
* Eppendorf 5410R refrigerated centrifuge with conical adapters | * Eppendorf 5410R refrigerated centrifuge with conical adapters | ||
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==Preparation== | ==Preparation== | ||
# Pick a 10 -12 large single colonies (2-3 mm dia) from your source plate and inoculate 500 ml of SOB medium in a 2 liter flask. | # Pick a 10 -12 large single colonies (2-3 mm dia) from your source plate and inoculate 500 ml of SOB medium in a 2 liter flask. | ||
# Grow to an OD of 0.6 with vigorous shaking (200-250 rpm) at 18 degrees | # Grow to an OD of 0.6 with vigorous shaking (200-250 rpm) at 18 degrees (important) | ||
# Prechill the centrifuge to 4 degrees | # Prechill the centrifuge to 4 degrees | ||
# Remove from the incubator and place on ice for 10 minutes | # Remove from the incubator and place on ice for 10 minutes | ||
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*[[Electrocompetent cells|Preparing electrocompetent cells]] | *[[Electrocompetent cells|Preparing electrocompetent cells]] | ||
*[[Electroporation]] | *[[Electroporation]] | ||
*[[TB buffer]] | |||
*[[Transforming chemically competent cells (Inoue)]] | |||
*[[Bacterial cell culture]] | *[[Bacterial cell culture]] | ||
Revision as of 18:19, 11 April 2006
Materials
- Plate of cells streaked for single colonies
- SOB media
- Ice
- TB buffer
- DMSO
- Dry Ice (or liquid nitrogen)
Glassware & equipment
- 2 liter erlenmeyer flask (no detergent residue, rinse with 70% ethanol and DI water)
- 220 ml conical centrifuge tubes BD 35 2075
- Eppendorf 5410R refrigerated centrifuge with conical adapters
Preparation
- Pick a 10 -12 large single colonies (2-3 mm dia) from your source plate and inoculate 500 ml of SOB medium in a 2 liter flask.
- Grow to an OD of 0.6 with vigorous shaking (200-250 rpm) at 18 degrees (important)
- Prechill the centrifuge to 4 degrees
- Remove from the incubator and place on ice for 10 minutes
- Transfer to two 220 ml centrifuge tubes and spin at 3220 x g for 10 minutes at 4 degrees
- Drain the medium and resuspend each pellet first in 5 ml of ice cold TB. Add an additional 75 ml of cold TB buffer and resuspend.
- Place on ice for 10 minutes
- Spin down as above.
- Resuspend each pellet in 20 ml of cold TB
- Add DMSO to a final concentration of 7%
- Incubate on ice for 10 minutes
- Dispense cells into pre-chilled tubes
- Freeze cells in a dry ice / ethanol bath and store at -80 degrees indefinitely
Related topics & references
- Preparing chemically competent cells
- Preparing TSS buffer
- Transforming chemically competent cells
- Preparing electrocompetent cells
- Electroporation
- TB buffer
- Transforming chemically competent cells (Inoue)
- Bacterial cell culture
Original protocol from Inoue et al. [1].