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 onevial 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
# 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 12:57, 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

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

store in freezer till ready for trasnformation

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