Team:BrownTwo/Protocols/compcells

From 2008.igem.org

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(New page: {{limitertemp}} =Making Ultracompetent Cells= *Transformation can be a major roadblock in assembling DNA constructs. Ligations can be hard to transform, especially with large inserts. D...)
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=Making Ultracompetent Cells=
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==Making Ultracompetent Cells==
*Transformation can be a major roadblock in assembling DNA constructs.  Ligations can be hard to transform, especially with large inserts.  DNA blotted on paper by the registry tends to be even harder.  We have had much better luck with the following protocol from Inoue than the standard CaCl2 method:
*Transformation can be a major roadblock in assembling DNA constructs.  Ligations can be hard to transform, especially with large inserts.  DNA blotted on paper by the registry tends to be even harder.  We have had much better luck with the following protocol from Inoue than the standard CaCl2 method:
 +
 +
==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==
 +
# 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.
 +
# Grow to  an OD of 0.6 with vigorous shaking (200-250 rpm) at 18 degrees (important).  This is slow -- approximately 35-40 hours.
 +
# 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.
 +
# While spinning, add 1.4 ml of DMSO to 18.6 ml of TB  (7% DMSO mixture)
 +
# Resuspend each pellet in 20 ml of cold TB-DMSO mixture
 +
# 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
 +
 +
==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--[[User:Melissali|mel]] 18:10, 14 June 2007 (EDT)
 +
 +
==Related topics & references==
 +
*[[Preparing chemically competent cells]]
 +
*[[TSS|Preparing TSS buffer]]
 +
*[[Transforming chemically competent cells]]''
 +
*[[Electrocompetent cells|Preparing electrocompetent cells]]
 +
*[[Electroporation]]
 +
*[[TB buffer]]
 +
*[[Transforming chemically competent cells (Inoue)]]
 +
*[[Bacterial cell culture]]
 +
 +
Original protocol from Inoue et al. <cite> Inoue90 </cite>.  Useful comments and speculation  about reducing agents in <cite>Hengen96</cite>.
 +
 +
<biblio>
 +
#Inoue90 pmid=2265755
 +
#Hengen96 pmid=8851666
 +
</biblio>
 +
 +
[[Category:Protocol]]
 +
[[Category:Escherichia coli]]

Revision as of 07:01, 28 October 2008



Contents

Making Ultracompetent Cells

  • Transformation can be a major roadblock in assembling DNA constructs. Ligations can be hard to transform, especially with large inserts. DNA blotted on paper by the registry tends to be even harder. We have had much better luck with the following protocol from Inoue than the standard CaCl2 method:

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. Inoue90 . Useful comments and speculation about reducing agents in Hengen96.

<biblio>

  1. Inoue90 pmid=2265755
  2. Hengen96 pmid=8851666

</biblio>

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