Paulsson:Electrocompetent E.coli

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Modified from Current Protocols in Molecular Biology

Electroporation with high voltage the most efficient method for transforming E. coli with plasmid DNA. The procedure described may be used to transform freshly prepared cells or to transform cells that have been previously grown and frozen. With freshly grown cells, it routinely gives more than 10(9) bacterial transformants per microgram of input plasmid DNA.

Materials

  • Ice cold means ice cold, no substitutions. Cold is the key to high transformation efficiency!!!!

Single colony of E. coli cells
LB medium
H2O, ice cold
10% glycerol, ice cold
500-ml centrifuge bottle, 50-ml narrow-bottom polypropylene tube, and microcentrifuge tubes, all chilled ice cold. Pre-chill pipets and pipet-tips by placing them in the -20 freezer.
Beckman J-6M centrifuge (or equivalent), pre-cooled
Beckman JS-4.2 rotor (or equivalent) and adaptors for 50-ml narrow-bottom tubes, pre-cooled

NOTE: All materials and reagents coming into contact with bacteria must be sterile. Duh!!!!


Prepare the cells

1. Inoculate a single colony of E. coli cells into 5 ml LB medium. Grow 5 hr to overnight at 37°C with moderate shaking (see UNIT 1.2).

2. Inoculate 2.5 ml of the culture into 500 ml LB medium in a sterile 2-liter flask. Grow at 37°C, shaking at 300 rpm, to an OD600 of ~0.5 to 0.7 (as measured in the Kirschner lab spectrophotometer, BioRad).

3. Chill cells in an ice-water bath 10 to 15 min. Transfer to a pre-chilled 500-ml centrifuge bottle (Silver Lab has a bunch)(don't fill bottle over 400ml - split 1 liter of culture into 3x 500ml bottles).

Cells should be kept at 2°C for all subsequent steps.

4. Centrifuge cells 20 min at 4200 rpm in Beckman J-4.2, 2°C.

5. Pour off supernatant and resuspend the pellet in 5 ml ice-cold water. Add 350-400 ml ice-cold water and mix well. Centrifuge cells as in step 4.

6. Pour off supernatant immediately and resuspend the pellet by swirling in remaining liquid.

Because the pellet is very loose, the supernatant must be poured off immediately. The pellet can be made tighter by substituting ice-cold sterile HEPES (1 mM, pH 7.0) for the ice-cold water in step 5.

7. Add another 350-400 ml ice-cold water, mix well, and centrifuge again as in step 4.

8. Pour off supernatant immediately and resuspend the pellet by swirling in remaining liquid.

9. Add 40 ml ice-cold 10% glycerol to the cells and mix well. Place suspension in a prechilled, narrow-bottom, 50-ml polypropylene tube, and centrifuge 10 min at 4200 rpm in Beckman JS-4.2 rotor and adaptors, 2°C.

10. Pour off supernatant. Estimate the pellet volume (usually ~500 µl from a 500-ml culture) and add an equal volume of ice-cold 10% glycerol to resuspend cells (on ice). Place 50- to 300-µl aliquots of cells into pre-chilled microcentrifuge tubes and freeze on dry ice (not in liquid nitrogen). Store at –80°C.

Prolonged incubation of cells in ice-water at all stages can increase transformation efficiency of some strains, such as BW313/P3 and MC1061/P3, >3-fold.

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