"
Team:Newcastle/Transformation of E. coli
From 2010.igem.org
(Difference between revisions)
PhilipHall (Talk | contribs) |
(→Protocol) |
||
| (2 intermediate revisions not shown) | |||
| Line 4: | Line 4: | ||
==Materials required== | ==Materials required== | ||
| - | * | + | * ''E. coli'' DH5α (200 µl) |
* Appropriate vector DNA | * Appropriate vector DNA | ||
* Heat block | * Heat block | ||
| Line 13: | Line 13: | ||
==Protocol== | ==Protocol== | ||
| - | # Thaw a 200 µl aliquot | + | # Thaw a 200 µl aliquot of ''E. coli'' DH4α and add the transforming DNA (10 ng of vector DNA in 10 µl). |
# Incubate on ice for 30 minutes. | # 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- | + | # Add 1 ml of LB broth and incubate the cells at 37°C for 1-1.5 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. | # Plate 200 µl of transformed ''E. coli'' onto 1.5% agar plate containing the appropriate selection markers. | ||
# Incubate the plates overnight at 37°C. | # Incubate the plates overnight at 37°C. | ||
Latest revision as of 14:37, 26 October 2010
| |||||||||||||
| |||||||||||||
Transformation of E. coli
Materials required
- E. coli DH5α (200 µl)
- Appropriate vector DNA
- Heat block
- Bucket of ice
- Pipettes
- Eppendorf tubes
- 1.5% agar plate containing appropriate antibiotics
Protocol
- Thaw a 200 µl aliquot of E. coli DH4α 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 1-1.5 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.
Go back to our Protocol List
   
|
    
|
