Purpose
Preparing E. coli chemically competent cells
Materials
- TBFI (pH 5.8; filter sterilized – keep at 4˚C when made to prevent precipitation)
| KOAc | 30mM |
| RbCl | 100mM |
| MnCl | 50mM |
| CaCl2 | 10mM |
When making the TBFI solution, in order to avoid precipitation of MnO2 (brown), dissolve everything except MnCl2 in ¾ of the volume, adjust pH to 6.0 with acetic acid add MnCl2 (dissolved in water), fill up volume to required amount and re-adjust pH to 5.8 if necessary. Avoid adding KOH (or any base!) to a solution containing Mn2+ salt.
- TBFII (pH 7.0; autoclaved)
| MOPS | 10mM |
| RbCl | 10mM |
| CaCl2 | 75mM |
| Glycerol | 15% |
Methods
- Streak E. coli strain from glycerol stock onto fresh LB agar plate with appropriate antibiotic. Grow at 37°C overnight.
- Select a single colony, use to inoculate a 5 mL LB culture (with appropriate antibiotics), incubate at 37°C overnight (shaking).
- Transfer 500 µl of the pre-culture into 200 mL pre-warmed, fresh LB (with appropriate antibiotics). Grow at 37°C (shaking) until OD600=0.45-0.55. From now on work on ice or at 4&Deg;C! Chill containers (5 minutes on ice) before transferring cells. Also treat cells very gently from now on.
- Aliquot culture in 4 x 50 mL Falcon tubes, and centrifuge at 3,000 rpm for 10 minutes (at 4°C). Discard supernatant. It is better to discard some cells with the supernatant than leave supernatant on the cells.
- Gently! Re-suspend pellets in 1 mL chilled TBFI and recombine pellets into one Flacon tube.
- Fill tube up to 15 mL with chilled TBFI and incubate on ice for 1 hour.
- Centrifuge at 3,000 rpm for 10 minutes and fully discard supernatant.
- Resuspend pellet in 4 mL (1/10 original culture volume) of cold TBFII.
- Aliquot competent cells (50 µl) and freeze immediately on dry ice. Store at -80°C.
Using E. coli chemically competent cells
- For each sample, thaw a vial of chemically competent cells on ice (10-15 minutes).
- Add 4 µl (~2-5 µg) of the DNA to each vial of thawed cells, mix gently by tapping. Incubate mixtures on ice for 30 minutes.
- Heat shock each vial of cells at 42°C for 30 seconds and immediately transfer to ice for 2 minutes.
- Add 250 µl of room temperature, fresh LB (or SOC) to the cells.
- Cap cells tightly and shake at 37°C, for 1 hour (expression of resistance gene).
- Plate cells (200, 100, 50 & 25 µl aliquots) on freshly made, pre-warmed LB + selective antibiotic plates. Incubate plates at 37°C overnight.
Original protocol by: C. Eshelman Date: 13 Sept 12
Protocol Updated by: ___ Date: ___
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