AU Biomaterials Design Lab:Protocols/Transformation Protocol
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# place reaction at 37 °C for 1 hour | # place reaction at 37 °C for 1 hour | ||
store in freezer till ready for trasnformation | store in freezer till ready for trasnformation | ||
| - | # prepare LB plates | + | # prepare LB plates |
| - | + | # Equilibrate water bath to 42 °C and place the LB plates at 37 °C | |
| - | + | # thaw one vial of cells on ice | |
| - | #Equilibrate water bath to 42 °C and place the LB plates at 37 °C | + | # add 10 ng of DNA in 5 μL to the cells (DO NOT MIX BY PIPETTING) |
| - | + | ||
| - | # thaw | + | |
| - | # add 10 ng of DNA in 5 μL to the cells ( DO NOT MIX BY PIPETTING) | + | |
# heat shock the cells by incubating in 42 °C water bath for exactly 30 s | # heat shock the cells by incubating in 42 °C water bath for exactly 30 s | ||
# place immediately on ice | # place immediately on ice | ||
| + | #add 250 μL od room temp SOC medium in the reaction vial while maintaining a sterile environment. | ||
| + | # shake at 225 rpm for 1 hour at 37 °C | ||
| + | # plate cells | ||
| + | ## for one plate use 50 μL of transformation reaction mixture | ||
| + | ## for a second plate use 200 μL of transformation reaction mixture | ||
| + | ## leftover transformation reaction mixture is stored at 4°C | ||
| + | # invert plates and incubate at 37 °C overnight | ||
Revision as of 13:57, 15 October 2012
Description
Transformation of PCR product into E. coli cells Transformation Manual
materials
Materials Supplied by the User
- Plasmid DNA (ready for transformation)
- 42°C water bath
- 37°C shaking and non-shaking incubator
- Ice bucket with ice
- Spectrophotometer to measure optical density of the cell cultures
- Microcentrifuge tube rack (optional)
Protocol
- to PCR product, add 1 μL DPN1
- place reaction at 37 °C for 1 hour
store in freezer till ready for trasnformation
- prepare LB plates
- Equilibrate water bath to 42 °C and place the LB plates at 37 °C
- thaw one vial of cells on ice
- add 10 ng of DNA in 5 μL to the cells (DO NOT MIX BY PIPETTING)
- heat shock the cells by incubating in 42 °C water bath for exactly 30 s
- place immediately on ice
- add 250 μL od room temp SOC medium in the reaction vial while maintaining a sterile environment.
- shake at 225 rpm for 1 hour at 37 °C
- plate cells
- for one plate use 50 μL of transformation reaction mixture
- for a second plate use 200 μL of transformation reaction mixture
- leftover transformation reaction mixture is stored at 4°C
- invert plates and incubate at 37 °C overnight
