McClean:Competent Cells: Difference between revisions

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{{back to protocols}}
{{back to protocols}}
This protocol is used for preparing competent cells for transformation.
*This protocol is used for preparing DH5 alpha competent cells for transformation with some modifications by Renee.
*The base protocol is the Zymo Mix & Go E. coli Transformation Kit & Buffer Set Protocol.


==Materials==
==Materials==
* Plate of cells streaked for single colonies
* LB Plate of DH5 alpha cells streaked for single colonies (This needs to be less than a week old.  For best results, store plate at room temp, not 4C.)
* LB
* [[SOB]] (not pHed!!!  The pH should be approx. 6.8-6.9  If the pH is at 7.5, the cells will grow too fast.)
* [[SOB]]
* [[LB]]
* Ice
* 2 buckets of Ice
* [[TB buffer]]
* Zymo Mix & Go kit
* DMSO
* Liquid nitrogen


 
==Glassware & Equipment==
==Glassware & equipment==
* Sterile culture tubes with lids
* 250 ml flask (no detergent residue, autoclaved)
* 2- 250 ml flasks with lids (no detergent residue, autoclaved)
* 1 liter flask (no detergent residue, autoclaved)
* 50 ml BD Falcon tubes
* 50 ml BD Falcon tubes  
* 15 ml BD Falcon tubes
* Eppendorf 5810R refrigerated centrifuge with conical adapters
* Sterile 1.5ml eppie tubes
* Beckman Coulter Allegra X14R refrigerated centrifuge with adapters


==Preparation==
==Preparation==
# 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.
# First thing in the morning, pick a single colony from your source plate and inoculate 5 ml of sterile LB medium in a sterile culture tube. Incubate the culture in 37°C for 4-5 hours with rotation.
# 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.  
# After the culture is incubated 4-5 hours, check the OD at 600nm.  If it is in log phase, use this culture to inoculate two 250ml flasks, each containing 50ml of SOB (not pHed). Calculate how much culture to put in the 50ml SOB to obtain an OD 600 of 0.01. Incubate both flasks at room temperature overnight with 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.
# The next morning, check OD600 of both flasks. Hopefully the flasks will be in log phase. (Depending on the temp of the room, this will usually take 16-19 hours.) If the flasks are between 0.4 and 0.6, put them on ice right away; otherwise, let them keep growing at room temp until they reach log phase.  I have been successful with an OD600 of 0.37.  You just get fewer cells.  I have also been successful with an OD600 of 0.68.  Too much more and it is a problem, though.
# Incubate the culture in ice-water bath for 10 minutes.   
# Incubate the culture on ice for 10 minutes.   
# Prechill the centrifuge to 4 degrees
# Pre-chill the centrifuge to 4 degrees.
# Split the culture to five 50ml tubes.
# Make 10ml each of 1X Wash Buffer and 1X Competent Buffer using the 2X stock solutions and Dilution Buffer.  EX. use 5ml of 2X Wash Buffer and 5ml Dilution Buffer. Mix well and keep on ice.
# Centrifuge at 3000g for 10 minutes.
# Pour the cultures into 50ml tubes.  Keep on ice.
# Drain the medium and store the open tubes on a stack of paper towels for 2 minutes.
# Centrifuge at 2500g for 5 minutes at 4C to gently pellet the cells.
# Resuspend each pellet in 40 ml of ice cold TB. Resuspend the cells by swirling rather than pipetting or vortexing.
# Decant the medium and drain the open tubes on a stack of paper towels for 10 seconds.  Get them back on ice.
# Centrifuge at 3000g for 10 minutes.
# Gently resuspend each pellet in 5 ml of ice cold 1X Wash Buffer. Resuspend the cells by pipetting gently with a large bore pipet (P1000), NOT vortexing. Keep on ice!!!
# Drain the medium and store the open tubes on a stack of paper towels for 2 minutes.
# Centrifuge at 2000g for 5 minutes at 4C.
# 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.
# Decant the medium as before. Get it back on ice.
# Add 1.5 ml of DMSO to the tube and mix.
# Add 5 ml ice-cold 1X Competent Buffer to each tube. Gently resuspend the cell pellets and put the tubes on ice.
# Incubate on ice for 10 minutes
# Aliquot the cells into 1.5ml eppie tubes on ice.  Each 50ml falcon tube is a separate batch of competent cells.  So this protocol makes 2 batches. Keep them on ice at all times. When finished aliquoting, put each batch in a labelled freezer box and place at -80 degrees to freeze.
# Dispense cells into tubes and drop the tubes to liquid nitrogen.  
# Store at -80 degrees.
# 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==
==Related topics & references==
*[[Preparing chemically competent cells]]
*[[Preparing chemically competent cells]]
*[[TSS|Preparing TSS buffer]]
*[[Transforming chemically competent cells]]''
*[[Transforming chemically competent cells]]''
*[[Electrocompetent cells|Preparing electrocompetent cells]]
*[[Electrocompetent cells|Preparing electrocompetent cells]]
*[[Electroporation]]
*[[Electroporation]]
*[[TB buffer]]
*[[Transforming chemically competent cells (Inoue)]]
*[[Transforming chemically competent cells (Inoue)]]
*[[Bacterial cell culture]]
*[[Bacterial cell culture]]

Latest revision as of 09:35, 9 May 2023

back to protocols
  • This protocol is used for preparing DH5 alpha competent cells for transformation with some modifications by Renee.
  • The base protocol is the Zymo Mix & Go E. coli Transformation Kit & Buffer Set Protocol.

Materials

  • LB Plate of DH5 alpha cells streaked for single colonies (This needs to be less than a week old. For best results, store plate at room temp, not 4C.)
  • SOB (not pHed!!! The pH should be approx. 6.8-6.9 If the pH is at 7.5, the cells will grow too fast.)
  • LB
  • 2 buckets of Ice
  • Zymo Mix & Go kit

Glassware & Equipment

  • Sterile culture tubes with lids
  • 2- 250 ml flasks with lids (no detergent residue, autoclaved)
  • 50 ml BD Falcon tubes
  • 15 ml BD Falcon tubes
  • Sterile 1.5ml eppie tubes
  • Beckman Coulter Allegra X14R refrigerated centrifuge with adapters

Preparation

  1. First thing in the morning, pick a single colony from your source plate and inoculate 5 ml of sterile LB medium in a sterile culture tube. Incubate the culture in 37°C for 4-5 hours with rotation.
  2. After the culture is incubated 4-5 hours, check the OD at 600nm. If it is in log phase, use this culture to inoculate two 250ml flasks, each containing 50ml of SOB (not pHed). Calculate how much culture to put in the 50ml SOB to obtain an OD 600 of 0.01. Incubate both flasks at room temperature overnight with shaking at 200rpm.
  3. The next morning, check OD600 of both flasks. Hopefully the flasks will be in log phase. (Depending on the temp of the room, this will usually take 16-19 hours.) If the flasks are between 0.4 and 0.6, put them on ice right away; otherwise, let them keep growing at room temp until they reach log phase. I have been successful with an OD600 of 0.37. You just get fewer cells. I have also been successful with an OD600 of 0.68. Too much more and it is a problem, though.
  4. Incubate the culture on ice for 10 minutes.
  5. Pre-chill the centrifuge to 4 degrees.
  6. Make 10ml each of 1X Wash Buffer and 1X Competent Buffer using the 2X stock solutions and Dilution Buffer. EX. use 5ml of 2X Wash Buffer and 5ml Dilution Buffer. Mix well and keep on ice.
  7. Pour the cultures into 50ml tubes. Keep on ice.
  8. Centrifuge at 2500g for 5 minutes at 4C to gently pellet the cells.
  9. Decant the medium and drain the open tubes on a stack of paper towels for 10 seconds. Get them back on ice.
  10. Gently resuspend each pellet in 5 ml of ice cold 1X Wash Buffer. Resuspend the cells by pipetting gently with a large bore pipet (P1000), NOT vortexing. Keep on ice!!!
  11. Centrifuge at 2000g for 5 minutes at 4C.
  12. Decant the medium as before. Get it back on ice.
  13. Add 5 ml ice-cold 1X Competent Buffer to each tube. Gently resuspend the cell pellets and put the tubes on ice.
  14. Aliquot the cells into 1.5ml eppie tubes on ice. Each 50ml falcon tube is a separate batch of competent cells. So this protocol makes 2 batches. Keep them on ice at all times. When finished aliquoting, put each batch in a labelled freezer box and place at -80 degrees to freeze.
  15. Store at -80 degrees.

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. High efficiency transformation of Escherichia coli with plasmids. Gene. 1990 Nov 30;96(1):23-8. DOI:10.1016/0378-1119(90)90336-p | PubMed ID:2265755 | HubMed [Inoue90]
  2. Hengen PN. Methods and reagents. preparing ultra-competent Escherichia coli. Trends Biochem Sci. 1996 Feb;21(2):75-6. PubMed ID:8851666 | HubMed [Hengen96]

All Medline abstracts: PubMed | HubMed

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