Difference between revisions of "AU Biomaterials Design Lab:Protocols/Transformation Protocol"

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(New page: ==Description== Transformation of PCR product into E. coli cells [http://tools.invitrogen.com/content/sfs/manuals/oneshotbl21_man.pdf Transformation Manual] ==materials== Materials Suppli...)
 
(Protocol)
 
(5 intermediate revisions by the same user not shown)
Line 1: Line 1:
 
==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==
 
Materials Supplied by the User
 
Materials Supplied by the User
Plasmid DNA (ready for transformation)
+
*Plasmid DNA (ready for transformation)
42°C water bath
+
*42°C water bath
37°C shaking and non-shaking incubator
+
*37°C shaking and non-shaking incubator
Ice bucket with ice
+
*Ice bucket with ice
Spectrophotometer to measure optical density of the
+
*Spectrophotometer to measure optical density of the cell cultures
cell cultures
+
*Microcentrifuge tube rack (optional)
Microcentrifuge tube rack (optional)
+
 
 
==Protocol==
 
==Protocol==
  
 
# 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 trasnformation
+
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 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 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 08:41, 24 October 2012

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