Wisconsin/Protocol:Transformation

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(Difference between revisions)
(Transforming Chemically Competent Cells)
(Transforming Chemically Competent Cells)
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#Grow 4 plates: 1 normal diluted, 1 normal concentrated, and 2 controls concentrated.
#Grow 4 plates: 1 normal diluted, 1 normal concentrated, and 2 controls concentrated.
#*Diluted: Spread 200uL onto plate
#*Diluted: Spread 200uL onto plate
-
#*Concentrated: Centrifuge all tubes for 5 minutes at 5000rpm first, then pippet everything except for 200uL onto plates.
+
#*Concentrated: Centrifuge tube for 5 minutes at 5000rpm first, then spread everything except for 200uL onto plates.
#Incubate plates for 16 hours at 37<sup>o</sup>C. Plates should face down to prevent condensation on surface.
#Incubate plates for 16 hours at 37<sup>o</sup>C. Plates should face down to prevent condensation on surface.

Revision as of 23:05, 5 July 2007

Transforming Chemically Competent Cells

  1. Thaw cells on ice
  2. Pippet 100uL of cells into three 1.5mL tube. One for transformation and the other two for control.
    • Normal: pippet 9uL of DNA into tube
    • Positive Control: pippet 2uL of pUC18 or pUC19 into tube
    • Negative Control: nothing
  3. Sit on ice for 30 minutes
  4. Heat shock in 42oC water bath for 30 seconds
  5. Incubate on ice for 2 minutes
  6. Transfer cells to new culture tubes (more O2 for growth) and add 900uL of SOC to each culture tube
  7. Incubate on shaker at 37oC for 1 hour
  8. Grow 4 plates: 1 normal diluted, 1 normal concentrated, and 2 controls concentrated.
    • Diluted: Spread 200uL onto plate
    • Concentrated: Centrifuge tube for 5 minutes at 5000rpm first, then spread everything except for 200uL onto plates.
  9. Incubate plates for 16 hours at 37oC. Plates should face down to prevent condensation on surface.