AU Biomaterials Design Lab:Protocols/Transformation Protocol: Difference between revisions
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==Description== | ==Description== | ||
Transformation of PCR product into E. coli cells | *Transformation of PCR product into E. coli cells | ||
[http://tools.invitrogen.com/content/sfs/manuals/oneshotbl21_man.pdf Transformation Manual] | *[http://tools.invitrogen.com/content/sfs/manuals/oneshotbl21_man.pdf Transformation Manual] | ||
==materials== | ==materials== | ||
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# to PCR product, add 1 μL DPN1 | # to PCR product, add 1 μL DPN1 | ||
# place reaction at 37 °C for 1 hour | # place reaction at 37 °C for 1 hour | ||
store in freezer till ready for | store in freezer till ready for transformation | ||
# prepare LB plates | # prepare LB plates | ||
https://www.eeb.ucla.edu/Faculty/Barber/Web%20Protocols/LB%20Agar%20Plates.pdf | |||
# Equilibrate water bath to 42 °C and place the LB plates at 37 °C | |||
#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 40 microliters of cells (DO NOT MIX BY PIPETTING) | |||
# thaw | |||
# add 10 ng of DNA in 5 μL to | |||
# 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 of 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 | |||
Latest revision as of 09:41, 24 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 transformation
- prepare LB plates
https://www.eeb.ucla.edu/Faculty/Barber/Web%20Protocols/LB%20Agar%20Plates.pdf
- 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 40 microliters of 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 of 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
