Team:Brown-Stanford/Lab/Protocols/NEB 5-alpha

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(→NEB 5-alpha High-Efficiency Transformation)
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#Mix the cells thoroughly by flicking the tube and inverting, then perform several 10-fold serial dilutions in SOC.
#Mix the cells thoroughly by flicking the tube and inverting, then perform several 10-fold serial dilutions in SOC.
#Spread 50-100 µl of each dilution onto a selection plate and incubate overnight at 37°C. Alternatively, incubate at 30°C for 24-36 hours or 25°C for 48 hours.
#Spread 50-100 µl of each dilution onto a selection plate and incubate overnight at 37°C. Alternatively, incubate at 30°C for 24-36 hours or 25°C for 48 hours.
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==Solutions needed==
 
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For C2987H: Thaw a tube of NEB 5-alpha Competent E. coli cells on ice for 10 minutes.
 
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For C2987I: Thaw a tube of NEB 5-alpha Competent E. coli cells on ice until the last ice crystals disappear. Mix gently and carefully pipette 50 µl of cells into a transformation tube on ice.
 
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Add 1-5 µl containing 1 pg-100 ng of plasmid DNA to the cell mixture. Carefully flick the tube 4-5 times to mix cells and DNA. Do not vortex.
 
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Place the mixture on ice for 30 minutes. Do not mix.
 
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Heat shock at exactly 42°C for exactly 30 seconds. Do not mix.
 
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Place on ice for 5 minutes. Do not mix.
 
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Pipette 950 µl of room temperature SOC into the mixture.
 
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Place at 37°C for 60 minutes. Shake vigorously (250 rpm) or rotate.
 
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Warm selection plates to 37°C.
 
-
Mix the cells thoroughly by flicking the tube and inverting, then perform several 10-fold serial dilutions in SOC.
 
-
Spread 50-100 µl of each dilution onto a selection plate and incubate overnight at 37°C. Alternatively, incubate at 30°C for 24-36 hours or 25°C for 48 hours.
 

Revision as of 06:37, 28 September 2011

NEB 5-alpha High-Efficiency Transformation

(Taken from [http://www.neb.com/nebecomm/products/protocol119.asp the New England BioLabs Protocol])

Overview

For C2987H, perform steps 1-7 in the tube provided.


Protocol

  1. For C2987H: Thaw a tube of NEB 5-alpha Competent E. coli cells on ice for 10 minutes.
  2. For C2987I: Thaw a tube of NEB 5-alpha Competent E. coli cells on ice until the last ice crystals disappear. Mix gently and carefully pipette 50 µl of cells into a transformation tube on ice.
  3. Add 1-5 µl containing 1 pg-100 ng of plasmid DNA to the cell mixture. Carefully flick the tube 4-5 times to mix cells and DNA. Do not vortex.
  4. Place the mixture on ice for 30 minutes. Do not mix.
  5. Heat shock at exactly 42°C for exactly 30 seconds. Do not mix.
  6. Place on ice for 5 minutes. Do not mix.
  7. Pipette 950 µl of room temperature SOC into the mixture.
  8. Place at 37°C for 60 minutes. Shake vigorously (250 rpm) or rotate.
  9. Warm selection plates to 37°C.
  10. Mix the cells thoroughly by flicking the tube and inverting, then perform several 10-fold serial dilutions in SOC.
  11. Spread 50-100 µl of each dilution onto a selection plate and incubate overnight at 37°C. Alternatively, incubate at 30°C for 24-36 hours or 25°C for 48 hours.
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