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...)
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==materials==
==materials==
Materials Supplied by the User
Materials Supplied by the User
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Plasmid DNA (ready for transformation)
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*Plasmid DNA (ready for transformation)
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42°C water bath
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*42°C water bath
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37°C shaking and non-shaking incubator
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*37°C shaking and non-shaking incubator
-
Ice bucket with ice
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*Ice bucket with ice
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Spectrophotometer to measure optical density of the
+
*Spectrophotometer to measure optical density of the cell cultures
-
cell cultures
+
*Microcentrifuge tube rack (optional)
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Microcentrifuge tube rack (optional)
+
 
==Protocol==
==Protocol==

Revision as of 12:51, 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
  1. thaw onevial of cells on ice
  2. add 10 ng of DNA in 5 μL to the cells ( DO NOT MIX BY PIPETTING)
  3. heat shock the cells by incubating in 42 °C water bath for exactly 30 s
  4. place immediately on ice
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