McClean:Competent Cells

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(New page: {{back to protocols}} This protocol is used for preparing competent cells for transformation. ==Materials== * Plate of cells streaked for single colonies * SOB * Ice * TB buffer *...)
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==Materials==
==Materials==
* Plate of cells streaked for single colonies
* Plate of cells streaked for single colonies
 +
* LB
* [[SOB]]
* [[SOB]]
* Ice
* Ice
* [[TB buffer]]
* [[TB buffer]]
* DMSO
* DMSO
-
* Dry Ice (or liquid nitrogen)
+
* Liquid nitrogen
-
*
+
 
==Glassware & equipment==
==Glassware & equipment==
-
* 2 liter erlenmeyer flask (no detergent residue, rinse with 70% ethanol and DI water)
+
* 250 ml flask (no detergent residue, autoclaved)
-
* 220 ml conical centrifuge tubes BD 35 2075
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* 1 liter flask (no detergent residue, autoclaved)
-
* Eppendorf 5410R refrigerated centrifuge with conical adapters
+
* 50 ml BD Falcon tubes
 +
* Eppendorf 5810R refrigerated centrifuge with conical adapters
==Preparation==
==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.
+
# Pick a single colonies (2-3 mm dia) from your source plate and inoculate 25 ml of sterile LB medium (do not use LB) in a 250mL flask. Incubate the culture in 37°C for 6-8 hours with vigorous shaking at 250rpm.
-
# Grow to an OD of 0.6 with vigorous shaking (200-250 rpm) at 18 degrees (important). This is slow -- approximately 35-40 hours.
+
# After the culture is incubated 6-8 hours, before you leave the lab,use this culture to inoculate three 1-liter flasks, each containing 250ml of SOB. The first flask receives 10ml of starter culture, the second receives 4ml and the third receives 2ml.Incubate all three flasks at room temperature overnight with moderate shaking at 200rpm.  
 +
# In the next morning, check OD600 with all three flasks.Pick the flask OD600 closest to 0.55. If the flask is a little over 0.55, put the flask in ice-water bath right away; otherwise keep monitor OD600 till it reaches 0.55.
 +
# Incubate the culture in ice-water bath for 10 minutes.  
# Prechill the centrifuge to 4 degrees
# Prechill the centrifuge to 4 degrees
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# Remove from the incubator and place on ice for 10 minutes
+
# Split the culture to five 50ml tubes.
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# Transfer to two 220 ml centrifuge tubes and spin at 3220 x g for 10 minutes at 4 degrees
+
# Centrifuge at 3000g for 10 minutes.
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# 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.
+
# Drain the medium and store the open tubes on a stack of paper towels for 2 minutes.
-
# Place on ice for 10 minutes
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# Resuspend each pellet in 40 ml of ice cold TB. Resuspend the cells by swirling rather than pipetting or vortexing.
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# Spin down as above.
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# Centrifuge at 3000g for 10 minutes.
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# While spinning, add 1.4 ml of DMSO to 18.6 ml of TB  (7% DMSO mixture)
+
# Drain the medium and store the open tubes on a stack of paper towels for 2 minutes.
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# Resuspend each pellet in 20 ml of cold TB-DMSO mixture
+
# Add 4 ml ice-cold TB buffer to each tube, resuspend the cell pellets then combine all of them to one tube and put the tube on ice.
 +
# Add 1.5 ml of DMSO to the tube and mix.
# Incubate on ice for 10 minutes
# Incubate on ice for 10 minutes
-
# Dispense cells into pre-chilled tubes
+
# Dispense cells into tubes and drop the tubes to liquid nitrogen.
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# Freeze cells in a dry ice / ethanol bath and store at -80 degrees indefinitely
+
# Store at -80 degrees.
==Thoughts on improvements==
==Thoughts on improvements==
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* The Hanahan protocol specifies dry pure DMSO, while Inoue says it doesn't make a difference.  Let's see.
* 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.
* 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==
==Related topics & references==

Revision as of 16:24, 31 October 2011

back to protocols

This protocol is used for preparing competent cells for transformation.

Contents

Materials

  • Plate of cells streaked for single colonies
  • LB
  • SOB
  • Ice
  • TB buffer
  • DMSO
  • Liquid nitrogen


Glassware & equipment

  • 250 ml flask (no detergent residue, autoclaved)
  • 1 liter flask (no detergent residue, autoclaved)
  • 50 ml BD Falcon tubes
  • Eppendorf 5810R refrigerated centrifuge with conical adapters

Preparation

  1. Pick a single colonies (2-3 mm dia) from your source plate and inoculate 25 ml of sterile LB medium (do not use LB) in a 250mL flask. Incubate the culture in 37°C for 6-8 hours with vigorous shaking at 250rpm.
  2. After the culture is incubated 6-8 hours, before you leave the lab,use this culture to inoculate three 1-liter flasks, each containing 250ml of SOB. The first flask receives 10ml of starter culture, the second receives 4ml and the third receives 2ml.Incubate all three flasks at room temperature overnight with moderate shaking at 200rpm.
  3. In the next morning, check OD600 with all three flasks.Pick the flask OD600 closest to 0.55. If the flask is a little over 0.55, put the flask in ice-water bath right away; otherwise keep monitor OD600 till it reaches 0.55.
  4. Incubate the culture in ice-water bath for 10 minutes.
  5. Prechill the centrifuge to 4 degrees
  6. Split the culture to five 50ml tubes.
  7. Centrifuge at 3000g for 10 minutes.
  8. Drain the medium and store the open tubes on a stack of paper towels for 2 minutes.
  9. Resuspend each pellet in 40 ml of ice cold TB. Resuspend the cells by swirling rather than pipetting or vortexing.
  10. Centrifuge at 3000g for 10 minutes.
  11. Drain the medium and store the open tubes on a stack of paper towels for 2 minutes.
  12. Add 4 ml ice-cold TB buffer to each tube, resuspend the cell pellets then combine all of them to one tube and put the tube on ice.
  13. Add 1.5 ml of DMSO to the tube and mix.
  14. Incubate on ice for 10 minutes
  15. Dispense cells into tubes and drop the tubes to liquid nitrogen.
  16. Store at -80 degrees.

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.

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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