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You can chose between chemically competent cells and electrocompetent cells. Andrew makes chemically competent cells for the lab to use. Several strains are kept in stock in the -80oC freezer.
Using Chemically Competent Cells
- Thaw frozen (-80oC) competent cells on ice.
- You REALLY dont want it to get too warm; add plasmids while it is still slushy.
- Add 1-10 uL DNA
- 10 uL if ligated plasmid. Use only 1 uL if regular plasmid from miniprep
- Incubate @ 42oC for 45 sec - 1 min
- Incubate on ice for 2 min
- Add 1 mL LB
- Sandy, Ceci, & Janet use 500 uL
- Incubate at 37oC for 45 min - 1 hr in eppendorf tubes
- Ideally shaking though it may not matter. You can tape your tubes to a rack in the shaker. Tape them well if you do -- they fly off!
- Pellet cells by centrifugation
- keep the ~100 uL droplet after you pour it off (Andrew)
- Plate 50-100 uL cells on LB (+antibiotic(s)) agar plate
- If you are worried about having a lawn, do one plate with more cells and dilute a fraction of the cells and plate a diluted aliquot.
Electrocompetent Cells (Incomplete)
- You can make your own electro competent cells.
- From Amada's past mentor in undergrad:
- For electrocompetent cells, I grow the cells overnight first, then inoculate again in the morning using O/N culture (the volume of new culture depends on how many tubes you want to prepare, let grow for about 3-4 h (OD 0.4-0.6 or so) and then centrifuge and wash 3 times with 10% glycerol (everything on ice). Finally I resuspend the pellet in a small volume (Example, if I do a 5 ml culture I try to resuspend the final pellet in no more than 0.2 or 0.3 ml of 10% glycerol). Then split in 1.5 ml centrifuge tubes (0.1 ml in each), freeze on dry ice-isopropanol and store at -80C.
- Use brown capped cuvettes for E. Coli. Wash, then sterilize with ethanol between uses. Do not autoclave: plastic will melt.