AU Biomaterials Design Lab:Protocols/Transformation Protocol

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

1. to PCR product, add 1 μL DPN1
2. place reaction at 37 °C for 1 hour

store in freezer till ready for transformation

1. prepare LB plates

https://www.eeb.ucla.edu/Faculty/Barber/Web%20Protocols/LB%20Agar%20Plates.pdf

1. Equilibrate water bath to 42 °C and place the LB plates at 37 °C
2. thaw one vial of cells on ice
3. add 10 ng of DNA in 5 μL to the cells (DO NOT MIX BY PIPETTING)
4. heat shock the cells by incubating in 42 °C water bath for exactly 30 s
5. place immediately on ice
6. add 250 μL od room temp SOC medium in the reaction vial while maintaining a sterile environment.
7. shake at 225 rpm for 1 hour at 37 °C
8. plate cells
   1. for one plate use 50 μL of transformation reaction mixture
   2. for a second plate use 200 μL of transformation reaction mixture
   3. leftover transformation reaction mixture is stored at 4°C
9. invert plates and incubate at 37 °C overnight