Wisconsin/Protocol:Transformation
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===Transforming Chemically Competent Cells=== | ===Transforming Chemically Competent Cells=== | ||
| - | #Thaw cells on ice | + | #Thaw competent cells on ice |
| - | # | + | #Chill three 1.5mL tubes in ice bucket. One for transformation and the other two for control. |
| - | #*Normal: pippet 9uL of | + | #*Normal: pippet 100uL of cells and 9uL of ligation reaction into tube |
| - | #*Positive Control: pippet 2uL of pUC18 or pUC19 into tube | + | #*Positive Control: pippet 100uL of cells and 2uL of pUC18 or pUC19 into tube |
| - | #*Negative Control: | + | #*Negative Control: pippet 100uL of cells |
#Sit on ice for 30 minutes | #Sit on ice for 30 minutes | ||
| - | #Heat shock in 42<sup>o</sup>C water bath for | + | #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 | ||
| - | # | + | #Add 200uL of SOC to each culture tube |
| - | #Incubate on shaker at 37<sup>o</sup>C for 1 hour | + | #Incubate on shaker (nutator) at 37<sup>o</sup>C for 1 hour |
#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 | + | #*Diluted: Spread 50-100uL onto plate |
| - | #*Concentrated: Centrifuge tube for | + | #*Concentrated: Centrifuge tube for 2-4 minutes at 5000rpm first, then spread everything except for 50-100uL 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. | ||
Latest revision as of 18:14, 11 July 2007
Transforming Chemically Competent Cells
- Thaw competent cells on ice
- 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
- Sit on ice for 30 minutes
- Heat shock in 42oC water bath for 90 seconds
- Incubate on ice for 2 minutes
- Add 200uL of SOC to each culture tube
- Incubate on shaker (nutator) at 37oC for 1 hour
- 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.
- Incubate plates for 16 hours at 37oC. Plates should face down to prevent condensation on surface.
