Difference between revisions of "Making chemically competent cells"
From SDMRC
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or use any commercially available premixed LB medium powder | or use any commercially available premixed LB medium powder | ||
| + | autoclave (using liquid setting) and store at room temperature until needed | ||
==== TS buffer ==== | ==== TS buffer ==== | ||
Latest revision as of 17:47, 20 January 2015
Contents
Making chemically competent cells
Method Summary
This method describes how to prepare chemically competent e.coli.
Materials
Luria broth (LB) medium
- 0.5g/L sodium chloride
- 10g/L tryptone
- 5g/L yeast extract
or use any commercially available premixed LB medium powder autoclave (using liquid setting) and store at room temperature until needed
TS buffer
- 1x Luria broth (LB) medium
- 10% PEG 8'000 (3'300 or similar works as well)
- 5% DMSO
- 10mM MnCl2
- 10mM MgSO4
sterile filter through 0.2μm filter. Possible to store at 4ºC.
Method
- Pick several colonies from starter plate
- Grow cells in LB medium to an OD600 of 0.3-0.6
- Pellet cells by centrifugation (4000rpm, 4ºC)
- Discard supernatant
- Resuspend cells on ice into 1/20 volume of TS buffer by pipetting or vortexing
- Incubate cells on ice for 10 minutes
- Aliquot into sterile tubes and snap freeze in liquid nitrogen
- Store cells until needed in -80ºC freezer
