Preparing chemically competent cells (Inoue)

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Current revision (12:09, 23 February 2009) (view source)
 
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{{back to protocols}}
==Materials==
==Materials==
* Plate of cells streaked for single colonies
* Plate of cells streaked for single colonies
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# Place on ice for 10 minutes
# Place on ice for 10 minutes
# Spin down as above.
# Spin down as above.
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# Resuspend each pellet in 20 ml of cold TB
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# While spinning, add 1.4 ml of DMSO to 18.6 ml of TB  (7% DMSO mixture)
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# Add DMSO to a final concentration of 7%
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# Resuspend each pellet in 20 ml of cold TB-DMSO mixture
# Incubate on ice for 10 minutes
# Incubate on ice for 10 minutes
# Dispense cells into pre-chilled tubes
# Dispense cells into pre-chilled tubes
# Freeze cells in a dry ice / ethanol bath and store at -80 degrees indefinitely
# Freeze cells in a dry ice / ethanol bath and store at -80 degrees indefinitely
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==Thoughts on improvements==
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* "Methods in Yeast Genetics" book (Amberg05) suggests growth the SOB + 300 mM NaCl
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* They also control pH at 7.5, which may be a major issue
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* Centrifuging in flat bottom centrifuge tubes may make pellet resuspension easier and less damaging
 +
* Length of time on ice prior to transformation may make a big difference
 +
* The Hanahan protocol specifies dry pure DMSO, while Inoue says it doesn't make a difference.  Let's see.
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* Warm plates for growing cells after transformation are claimed to be 2x to 4x more efficient.
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* My lab uses LB for instead of SOB media...it seems to work fine for them--[[User:Melissali|mel]] 18:10, 14 June 2007 (EDT)
==Related topics & references==
==Related topics & references==
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*[[Bacterial cell culture]]
*[[Bacterial cell culture]]
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Original protocol from Inoue et al. <cite> Inoue90 </cite>.
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Original protocol from Inoue et al. <cite> Inoue90 </cite>.  Useful comments and speculation  about reducing agents in <cite>Hengen96</cite>.
<biblio>
<biblio>
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</biblio>
</biblio>
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[[Category:E.Coli Protocol]]
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[[Category:Protocol]]
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[[Category:Escherichia coli]]

Current revision

back to protocols

Contents

Materials

  • Plate of cells streaked for single colonies
  • SOB
  • 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

  1. Pick a 10 - 12 large single colonies (2-3 mm dia) from your source plate and inoculate 500 ml of sterile SOB medium (do not use LB) in a 2 liter flask. Save some medium as an OD blank.
  2. Grow to an OD of 0.6 with vigorous shaking (200-250 rpm) at 18 degrees (important). This is slow -- approximately 35-40 hours.
  3. Prechill the centrifuge to 4 degrees
  4. Remove from the incubator and place on ice for 10 minutes
  5. Transfer to two 220 ml centrifuge tubes and spin at 3220 x g for 10 minutes at 4 degrees
  6. 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.
  7. Place on ice for 10 minutes
  8. Spin down as above.
  9. While spinning, add 1.4 ml of DMSO to 18.6 ml of TB (7% DMSO mixture)
  10. Resuspend each pellet in 20 ml of cold TB-DMSO mixture
  11. Incubate on ice for 10 minutes
  12. Dispense cells into pre-chilled tubes
  13. Freeze cells in a dry ice / ethanol bath and store at -80 degrees indefinitely

Thoughts on improvements

  • "Methods in Yeast Genetics" book (Amberg05) suggests growth the SOB + 300 mM NaCl
  • They also control pH at 7.5, which may be a major issue
  • Centrifuging in flat bottom centrifuge tubes may make pellet resuspension easier and less damaging
  • Length of time on ice prior to transformation may make a big difference
  • The Hanahan protocol specifies dry pure DMSO, while Inoue says it doesn't make a difference. Let's see.
  • Warm plates for growing cells after transformation are claimed to be 2x to 4x more efficient.
  • My lab uses LB for instead of SOB media...it seems to work fine for them--mel 18:10, 14 June 2007 (EDT)

Related topics & references

Original protocol from Inoue et al. [1]. Useful comments and speculation about reducing agents in [2].

  1. Inoue H, Nojima H, and Okayama H. . pmid:2265755. PubMed HubMed [Inoue90]
  2. Hengen PN. . pmid:8851666. PubMed HubMed [Hengen96]
All Medline abstracts: PubMed HubMed
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