Wisconsin/Protocol:Transformation

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< Wisconsin(Difference between revisions)
(→Transforming Chemically Competent Cells)
(→Transforming Chemically Competent Cells)
 
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===Transforming Chemically Competent Cells===
===Transforming Chemically Competent Cells===
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#Thaw cells on ice
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#Thaw competent cells on ice
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#Pippet 100uL of cells into three 1.5mL tube. One for transformation and the other two for control.
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#Chill three 1.5mL tubes in ice bucket. One for transformation and the other two for control.
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#*Normal: pippet 9uL of DNA into tube
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#*Normal: pippet 100uL of cells and 9uL of ligation reaction into tube
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#*Positive Control: pippet 2uL of pUC18 or pUC19 into tube
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#*Positive Control: pippet 100uL of cells and 2uL of pUC18 or pUC19 into tube
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#*Negative Control: nothing
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#*Negative Control: pippet 100uL of cells
#Sit on ice for 30 minutes
#Sit on ice for 30 minutes
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#Heat shock in 42<sup>o</sup>C water bath for 30 seconds
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#Heat shock in 42<sup>o</sup>C water bath for 90 seconds
#Incubate on ice for 2 minutes
#Incubate on ice for 2 minutes
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#Transfer cells to new culture tubes (more O<sub>2</sub> for growth) and add 900uL of SOC to each culture tube
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#Add 200uL of SOC to each culture tube
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#Incubate on shaker at 37<sup>o</sup>C for 1 hour
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#Incubate on shaker (nutator) at 37<sup>o</sup>C for 1 hour
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#Spread ??? uL onto plate... centrifuge first???
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#Grow 4 plates: 1 normal diluted, 1 normal concentrated, and 2 controls concentrated.
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#Incubate plates for 16 hours at 37<sup>o</sup>C. Put plates upside down to prevent condensation on surface.
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#*Diluted: Spread 50-100uL onto plate
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#*Concentrated: Centrifuge tube for 2-4 minutes at 5000rpm first, then spread everything except for 50-100uL onto plates.
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#Incubate plates for 16 hours at 37<sup>o</sup>C. Plates should face down to prevent condensation on surface.

Latest revision as of 18:14, 11 July 2007

Transforming Chemically Competent Cells

  1. Thaw competent cells on ice
  2. Chill three 1.5mL tubes in ice bucket. One for transformation and the other two for control.
    • Normal: pippet 100uL of cells and 9uL of ligation reaction into tube
    • Positive Control: pippet 100uL of cells and 2uL of pUC18 or pUC19 into tube
    • Negative Control: pippet 100uL of cells
  3. Sit on ice for 30 minutes
  4. Heat shock in 42oC water bath for 90 seconds
  5. Incubate on ice for 2 minutes
  6. Add 200uL of SOC to each culture tube
  7. Incubate on shaker (nutator) at 37oC for 1 hour
  8. Grow 4 plates: 1 normal diluted, 1 normal concentrated, and 2 controls concentrated.
    • Diluted: Spread 50-100uL onto plate
    • Concentrated: Centrifuge tube for 2-4 minutes at 5000rpm first, then spread everything except for 50-100uL onto plates.
  9. Incubate plates for 16 hours at 37oC. Plates should face down to prevent condensation on surface.
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