Wisconsin/Protocol:Transformation

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(Difference between revisions)
(Transforming Chemically Competent Cells)
Line 8: Line 8:
#Heat shock in 42<sup>o</sup>C water bath for 30 seconds
#Heat shock in 42<sup>o</sup>C water bath for 30 seconds
#Incubate on ice for 2 minutes
#Incubate on ice for 2 minutes
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#Add 900uL of SOC to each tube
+
#Transfer cells to new culture tubes (more O<sub>2</sub> for growth) and add 900uL of SOC to each culture tube
#Incubate on shaker at 37<sup>o</sup>C for 1 hour
#Incubate on shaker at 37<sup>o</sup>C for 1 hour
#Spread ??? uL onto plate... centrifuge first???
#Spread ??? uL onto plate... centrifuge first???
#Incubate plates for 16 hours at 37<sup>o</sup>C. Put plates upside down to prevent condensation on surface.
#Incubate plates for 16 hours at 37<sup>o</sup>C. Put plates upside down to prevent condensation on surface.

Revision as of 22:41, 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. Spread ??? uL onto plate... centrifuge first???
  9. Incubate plates for 16 hours at 37oC. Put plates upside down to prevent condensation on surface.