Paulsson:Electrocompetent E.coli v2

Modified from version 1: Larger volumes; spins in Corning conical centrifuge bottles; slower spins

Materials
All containers and solutions should be pre-chilled in ice-water baths, and pipets pre-chilled in a freezer when practical Performing resuspension steps in the cold room can't hurt... I usually just do the last transfer to microfuge tubes in the cold room as other steps can be done in bottles in ice-water bath placed on the bench.
 * Ice cold means ice cold, no exceptions. Cold is the key to high transformation efficiency!!!!

Freshly streaked E.coli strain (e.g. MC1061 = PMB12) 3L LB medium 5L H2O, ice cold 500ml 10% glycerol, ice cold 6x 500-ml centrifuge bottles 8x 50-ml narrow-bottom polypropylene tube ("Falcon tube") ~65x microcentrifuge tubes 18x 10ml pipets 10x 25ml pipets 4x 500ml graduated cylinders Beckman J-6M centrifuge (or equivalent), pre-cooled to 2°C Beckman JS-4.2 rotor (or equivalent) and adapters, 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 40 ml LB medium. Grow overnight at 37°C with moderate shaking.

2. Inoculate 15 ml of the culture into 3L LB medium, split into two 4L flasks. Grow at 37°C, shaking at ~200 rpm, to an OD600 of ~0.5 to 0.7 (as measured in the Kirschner lab spectrophotometer, BioRad). MC1061 grows slowly, takes 4 hours.

3. Chill cells in an ice-water bath 20 to 30 min; swirl occasionally to mix (every ~5 min). Transfer to pre-chilled 500-ml centrifuge bottles.

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

4. Centrifuge cells 20 min at 3400 rpm in Beckman J-4.2, 2°C.

5. Pour off supernatant and add 10 ml ice-cold water to each bottle. Vortex briefly (~5s) and return to ice-water bath. Do for each bottle, then repeat. Using a pre-chilled pipet, resuspend the pellet, then add 400 ml ice-cold water and mix well.

6. Centrifuge cells as in step 4, increasing the speed to 3600 rpm.

7. Pour off supernatant, resuspend the pellet as above, and add another 380 ml ice-cold water, mix well, and

8. Centrifuge again as in step 4, increasing the speed to 3900 rpm.

8. Pour off supernatant as bottles are removed from centrifuge. Resuspend the pellet with 10 ml 10% glycerol. Transfer to 50 ml conicals. Rinse the bottle with an additional 30 ml 10% glycerol and transfer to the same conical tube, mixing.

9. Centrifuge 10 min at 3900 rpm in Beckman JS-4.2 rotor and adaptors, 2°C.

10. Aspirate supernatant carefully (pellet not entirely stable). Estimate the pellet volume (~500 µl from 500 ml culture) and add an equal volume of ice-cold 10% glycerol. Resuspend cells on ice using wide-mouth P1000 tips (cut ~5mm from tip). Place ~105 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.