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Team:Newcastle/Transformation of E. coli
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==Procedures== | ==Procedures== | ||
| - | # Thaw a 200 µl aliquot of the desired strain of ''E. coli'' and add the transforming DNA (10 ng of vector DNA in 10 µl). # Incubate on ice for 30 minutes. | + | # Thaw a 200 µl aliquot of the desired strain of ''E. coli'' and add the transforming DNA (10 ng of vector DNA in 10 µl). |
| + | # Incubate on ice for 30 minutes. | ||
# Heat-shock the cells for 120 seconds at 42°C, and place on ice again for 3-4 minutes. | # Heat-shock the cells for 120 seconds at 42°C, and place on ice again for 3-4 minutes. | ||
# Add 1 ml of LB broth and incubate the cells at 37°C for 1-15 hr in a water bath with gentle shaking. | # Add 1 ml of LB broth and incubate the cells at 37°C for 1-15 hr in a water bath with gentle shaking. | ||
Revision as of 14:36, 29 July 2010
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Transformation of E. coli
Materials required
- Appropriate E. coli strains (200 µl)
- Appropriate vector DNA
- Heat block
- Bucket of ice
- Pipettes
- Eppendorf tubes
- 1.5% agar plate containing appropriate antibiotics
Procedures
- Thaw a 200 µl aliquot of the desired strain of E. coli and add the transforming DNA (10 ng of vector DNA in 10 µl).
- Incubate on ice for 30 minutes.
- Heat-shock the cells for 120 seconds at 42°C, and place on ice again for 3-4 minutes.
- Add 1 ml of LB broth and incubate the cells at 37°C for 1-15 hr in a water bath with gentle shaking.
- Plate 200 µl of transformed E. coli onto 1.5% agar plate containing the appropriate selection markers.
- Incubate the plates overnight at 37°C.
   
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