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Team:EPF-Lausanne/Protocols/Competent cells. Protocol II
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* Plate of cells streaked for single colonies | * Plate of cells streaked for single colonies | ||
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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. | ||
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Latest revision as of 08:48, 15 July 2011
Competent cells preparation (by Inoue)
Back to protocols.Contents |
Materials
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.
