AU Biomaterials Design Lab:Protocols/Transformation Protocol: Difference between revisions
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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 10:57, 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