"
Team:Newcastle/Transformation of E. coli
From 2010.igem.org
(Difference between revisions)
(→Procedures) |
|||
| Line 13: | Line 13: | ||
==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 for | + | # 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, 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. | ||
| - | # Plate | + | # Plate 200 µl of transformed ''E. coli'' onto 1.5% agar plate containing the appropriate selection markers. |
| - | # Incubate plates overnight at 37°C. | + | # Incubate the plates overnight at 37°C. |
{{Team:Newcastle/footer}} | {{Team:Newcastle/footer}} | ||
Revision as of 14:35, 29 July 2010
| |||||||||||||
| |||||||||||||
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.
   
|
    
|
