6. Preparing and using competent E. coli cells
Competent cells are prepared by the method of Chung et al (1989):
Chung, C.T., Niemela, S.L., and Miller, R.H. 1989. One-step preparation of competent Escherichia coli – transformation and storage of bacterial cells in the same solution. Proc. Natl. Acad. Sci. USA 86, 2172-2175.
Note: This procedure is quick and easy and gives reliable results in my hands. However, a procedure for generating cells of much higher competence is given on the main Openwetware protocols site, and we may change to this in due course.
Transformation and Storage Solution (1 x TSS)
To make 24 ml TSS, mix the following:
- 17 ml LB broth
- 5 ml 40% w/v PEG 3350
- 1 ml 1 M MgCl2
- 1 ml DMSO
All components apart from DMSO should be sterilised by autoclaving prior to mixing. Do not autoclave the TSS after it has been made, as this causes precipitation. Store the TSS at 4˚C.
- 1. Streak E. coli from a -80˚C stock to L agar (with chloramphenicol if pLysS present). Incubate at 37˚C overnight. You can leave the plate for several days at room temperature before using it, if required.
- 2. Inoculate 50 ml LB (+ chloramphenicol if pLysS) in a 250 ml flask with a loopful of cell material and incubate at 37˚C with shaking for about 4 to 4.5 hours, to OD600 of about 0.2 to 0.5 or so (the exact value is not critical).
- 3. Chill the flask in an ice-water slurry to bring the temperature down quickly, then place it on ice. Also place 12 or 24 x 1.5 ml microcentrifuge tubes on ice.
Note: the cells must be kept cold from this point on!
- 4. For each tube of competent cells you want to prepare, put 1 ml of culture in a pre-chilled microcentrifuge tube. Spin down the cells, preferably in the cold room (microcentrifuge at 'high' 'speed for about 4 to 5 minutes).
- 5. Remove the supernatant to a waste bottle. Add 100 microlitres of 1 x TSS to each tube and resuspend the cells by pipetting up and down in 100 ul of ice-cold 1 x TSS.
Note: the waste bottle should be placed out for autoclaving. All waste solutions potentially containing live cultures MUST be autoclaved prior to disposal.
- 6. Leave on ice for about 30 minutes (according to the original publication, the exact amount of time left on ice is not critical).
- 7. Either transform the cells immediately or place at -80˚C for future use. I do not routinely snap freeze the cells in liquid nitrogen; generally I just place them in the -80˚C freezer. There is some loss of comptence on freezing, but the cells should still be adequate for routine transformations. Where high competence is required, it may be advantageous to prepare cells immediately beforehand and use them without freezing.
Alternative procedure for preparing larger batches
- 4. After the culture has been chilled, spin down the whole 50 ml in a sterile Falcon tube.
- 5. Pour off the supernatant and resuspend the cell pellet in 5 ml ice-cold 1 x TSS (or altenatively, add to the pellet 3.4 ml chilled LB, 1 ml 40% PEG, 0.2 ml 1 M MgCl2, and 0.2 ml DMSO, swirl, and resuspend the cells). Keep the tube cold. Transfer 0.1 ml of the suspension into each of about 51 pre-chilled 1.5 ml microcentrifuge tubes.
- 5b. In order to save space and reduce the amount of pipetting you need to do, you can put 200 microlitres in each tube rather than 100 microlitres. This gives you about 25 tubes from a 50 ml culture. When you come to use the cells, after thawing them, you can do 2 transformations from one tube by transferring 100 microlitres to a fresh pre-chilled tube before adding the DNA. If you only want to do one transformation, just use the tube as usual but add 800 microlitres of LB rather than 900 microlitres.
- 6. Continue from step 6 above.
- The advantage of this version is that you avoid the repetitive strain injury associated with resuspending a large number of pellets. The procedure seems to work just as well as the original version.
- 1. If using frozen cells, remove one tube of cells from the freezer for each transformation. Place the cells in an ice-water slurry for a few minuts to thaw, then transfer them to ice. The cells must be kept cold.
- 2. Add DNA (generally 1 microlitre of supercoiled plasmid DNA or 5 microlitres of ligation) and mix gently. Incubate on ice for 30 to 60 minutes.
- 2b. NEW (5 Oct 09): According to the original paper, a heat shock is not necessary, but we have recently been adding a heat shock step at this stage and it seems to make a big difference. My practice is to heat shock the cells in a 42 C waterbath for 90 seconds, then return them to ice for 90 seconds before adding the LB recovery medium (step 3).
- 3. Add 900 microlitres of LB and mix by inversion. Incubate at 37˚C for 45 to 60 min.
- 2b and 3b: if you are using tubes with 200 microlitres of cells rather than 100 microlitres, you can do 2 transformations from one tube by transferring 100 microlitres to a fresh pre-chilled tube before adding the DNA. If you only want to do one transformation, just use the tube as usual but add 800 microlitres of LB rather than 900 microlitres.
- 4. Plate 100 microlitres of cells to a plate with appropriate antibiotics. This should be OK if using supercoiled plasmid DNA. Transformation efficiency with relaxed DNA from a ligation is much lower. In this case, you should also gently spin down the remaining 900 microlitres of cells (microcentrifuge, 8 000 rpm for 4 minutes), remove all of the supernatant except for 100 microlitres, and then resuspend the cells in the remaining 100 microlitres of liquid and plate them on a second plate.
Safety notes:None of the chemicals used in these procedures is significantly toxic (with the exception of certain antibiotics). Always wear gloves when transferring items to or from the -80˚C freezers (bare skin may stick to the metal due to the extreme cold). Ensure that all liquids which may contain live bacteria are autoclaved prior to disposal.