AU Biomaterials Design Lab:Protocols/Transformation Protocol

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Current revision (12:41, 24 October 2012) (view source)
(Protocol)
 
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# 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
# thaw one vial of cells on ice
-
# add 10 ng of DNA in 5 μL to the cells (DO NOT MIX BY PIPETTING)
+
# 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
# 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.
+
#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
# shake at 225 rpm for 1 hour at 37 °C
# plate cells
# plate cells

Current revision

Description

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 40 microliters of 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 of 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
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