Making chemically competent cells

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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

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

  1. Pick several colonies from starter plate
  2. Grow cells in LB medium to an OD600 of 0.3-0.6
  3. Pellet cells by centrifugation (4000rpm, 4ºC)
  4. Discard supernatant
  5. Resuspend cells on ice into 1/20 volume of TS buffer by pipetting or vortexing
  6. Incubate cells on ice for 10 minutes
  7. Aliquot into sterile tubes and snap freeze in liquid nitrogen
  8. Store cells until needed in -80ºC freezer
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