Lidstrom:Transformation

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Revision as of 15:00, 30 April 2012 by Janet B. Matsen (Talk | contribs)
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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:
      • Grow the cells overnight
      • Inoculate from this the next morning: generally use 200 uL/50 mL
      • Let grow for about 3-4 h (OD 0.4-0.6 or so), then centrifuge and wash 3 times with 10% glycerol (everything on ice).
      • 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.
    • Janet called BioRad on 4/2012 and they said ethanol sterilization is fine, but do not autoclave or the plastic will melt. They want you to buy new ones every time instead, but that is silly.
  • To transform cells:
    • use ice cold electrocompetent cells
    • mix in 1-2 uL DNA; gently mix to prevent shear stress on cells & DNA
    • add ~950 uL SOC media
    • set voltage to either 1.25 kV (1mm (brown cap) cuvettes) or 2.5 kV (2mm (green cap) cuvettes).
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