BMBMethod:CompetentCells
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Preparation of Chemically Competent Cells
Adapted from Inoue et al. 1990
Capable of ~3x109 cfu/ug
- Inoculate E. coli into 150mL SOB or LB media
- Grow between 18°C and 25°C until an OD600 of 0.5-0.6 is reached (overnight more convenient)
- At 18°C, it can certainly take 48+ hrs.
- Cool flask of cells on ice for 10 minutes
- Pellet cells by centrifugation at 4°C
- Gently resuspend pellet in 40mL of ice-cold TB
- Incubate cells on ice for 10 min
- Pellet cells by centrifugation at 4°C
- Resuspend cells in 5.58 mL of TB
- Add DMSO to final concentration of 7% (420 ul; 6mL final vol)
- Dispense into sterile microfuge tubes in 50-200ul aliquots
- Immediately snap freeze in liquid nitrogen
- Store in -80°C freezer until time to use
Transformation Buffer (TB) Recipe (50mL)
Reagent | Amount | Final Concentration |
---|---|---|
PIPES | xxxg | 10mM |
CaCl2 | xxxg | 15mM |
KCl | xxxg | 250mM |
pH | 6.7 (w/KOH) | pH BEFORE adding MnCl2 |
MnCl2 | xxxg | 55mM |
References
Inoue, H., Nojima, H., & Okayama, H. (1990). High efficiency transformation of Escherichia coli with plasmids. Gene, 96(1), 23–8. Retrieved from http://www.ncbi.nlm.nih.gov/pubmed/2265755