Wisconsin/Protocol:Transformation
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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 | + | #*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
- Thaw cells on ice
- 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
- Sit on ice for 30 minutes
- Heat shock in 42oC water bath for 30 seconds
- Incubate on ice for 2 minutes
- Transfer cells to new culture tubes (more O2 for growth) and add 900uL of SOC to each culture tube
- Incubate on shaker at 37oC for 1 hour
- 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.
- Incubate plates for 16 hours at 37oC. Plates should face down to prevent condensation on surface.
