Lidstrom:Transformation

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(Electrocompetent Cells (Incomplete))
(Electrocompetent Cells (Incomplete))
Line 33: Line 33:
**use ice cold electrocompetent cells
**use ice cold electrocompetent cells
**mix in 1-2 uL DNA; gently mix to prevent shear stress on cells & DNA
**mix in 1-2 uL DNA; gently mix to prevent shear stress on cells & DNA
-
**add ~950 uL [[SOC| SOC media]]
+
**transfer mixture to sterile cuvette
 +
***make sure there are no bubbles or it could arc (bad for your cells)
**set voltage to either 1.25 kV (1mm (brown cap) cuvettes) or 2.5 kV (2mm (green cap) cuvettes).
**set voltage to either 1.25 kV (1mm (brown cap) cuvettes) or 2.5 kV (2mm (green cap) cuvettes).
 +
**shock them by pressing both buttons
 +
**as fast as possible, add ~950 uL [[SOC| SOC media]] & transfer to ice-cold sterilized cuvette.  Fast --> less cells die.
 +
**Transfer SOC-cell mixture to chilled eppendorf tube.
 +
**Chill sample on ice for 2 mins to permit the cells to recover.
 +
**Transfer eppendorf tube to 37°C incubator and shake to promote aeration. Incubate for 1 hr to permit expression of antibiotic resistance gene.
 +
**Plate transformation onto prewarmed LB-agar plate supplemented with appropriate antibiotic. I generally plate 200μL but appropriate plating volume depends on efficiency of the transformation.
 +
**Incubate plate overnight at 37°C.
 +
**Leave remaining SOC-cell mixture on the benchtop overnight.
 +
**If you don't have any transformants, plate the rest of the transformation in the morning.

Revision as of 16:04, 30 April 2012

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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
    • transfer mixture to sterile cuvette
      • make sure there are no bubbles or it could arc (bad for your cells)
    • set voltage to either 1.25 kV (1mm (brown cap) cuvettes) or 2.5 kV (2mm (green cap) cuvettes).
    • shock them by pressing both buttons
    • as fast as possible, add ~950 uL SOC media & transfer to ice-cold sterilized cuvette. Fast --> less cells die.
    • Transfer SOC-cell mixture to chilled eppendorf tube.
    • Chill sample on ice for 2 mins to permit the cells to recover.
    • Transfer eppendorf tube to 37°C incubator and shake to promote aeration. Incubate for 1 hr to permit expression of antibiotic resistance gene.
    • Plate transformation onto prewarmed LB-agar plate supplemented with appropriate antibiotic. I generally plate 200μL but appropriate plating volume depends on efficiency of the transformation.
    • Incubate plate overnight at 37°C.
    • Leave remaining SOC-cell mixture on the benchtop overnight.
    • If you don't have any transformants, plate the rest of the transformation in the morning.
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