McClean:Competent Cells: Difference between revisions
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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 | ||
* | * Liquid nitrogen | ||
==Glassware & equipment== | ==Glassware & equipment== | ||
* | * 250 ml flask (no detergent residue, autoclaved) | ||
* | * 1 liter flask (no detergent residue, autoclaved) | ||
* Eppendorf | * 50 ml BD Falcon tubes | ||
* Eppendorf 5810R refrigerated centrifuge with conical adapters | |||
==Preparation== | ==Preparation== | ||
# Pick a | # 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. | ||
# | # 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 | ||
# | # Split the culture to five 50ml tubes. | ||
# | # Centrifuge at 3000g for 10 minutes. | ||
# Drain the medium and | # Drain the medium and store the open tubes on a stack of paper towels for 2 minutes. | ||
# | # Resuspend each pellet in 40 ml of ice cold TB. Resuspend the cells by swirling rather than pipetting or vortexing. | ||
# | # Centrifuge at 3000g for 10 minutes. | ||
# | # Drain the medium and store the open tubes on a stack of paper towels for 2 minutes. | ||
# | # 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 | # Dispense cells into tubes and drop the tubes to liquid nitrogen. | ||
# | # Store at -80 degrees. | ||
==Thoughts on improvements== | ==Thoughts on improvements== | ||
| Line 38: | Line 43: | ||
* 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. | ||
==Related topics & references== | ==Related topics & references== | ||
Revision as of 13:24, 31 October 2011
| back to protocols | ||
This protocol is used for preparing competent cells for transformation.
Materials
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
- 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.
- 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
- Split the culture to five 50ml tubes.
- Centrifuge at 3000g for 10 minutes.
- Drain the medium and store the open tubes on a stack of paper towels for 2 minutes.
- Resuspend each pellet in 40 ml of ice cold TB. Resuspend the cells by swirling rather than pipetting or vortexing.
- Centrifuge at 3000g for 10 minutes.
- Drain the medium and store the open tubes on a stack of paper towels for 2 minutes.
- 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
- Dispense cells into tubes and drop the tubes to liquid nitrogen.
- 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
- Preparing chemically competent cells
- Preparing TSS buffer
- Transforming chemically competent cells
- Preparing electrocompetent cells
- Electroporation
- TB buffer
- Transforming chemically competent cells (Inoue)
- Bacterial cell culture
Original protocol from Inoue et al. [1]. Useful comments and speculation about reducing agents in [2].
- 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 |
- Hengen PN. Methods and reagents. preparing ultra-competent Escherichia coli. Trends Biochem Sci. 1996 Feb;21(2):75-6.
