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

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("'NEB 5-alpha High-Efficiency Transformation)
(NEB 5-alpha High-Efficiency Transformation)
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=== NEB 5-alpha High-Efficiency Transformation ===
=== NEB 5-alpha High-Efficiency Transformation ===
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Taken from [http://www.neb.com/nebecomm/products/protocol119.asp the New England BioLabs Protocol]
+
(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.
 +
 
 +
 
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==Protocol==
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#For C2987H: Thaw a tube of NEB 5-alpha Competent E. coli cells on ice for 10 minutes.
 +
#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.
 +
#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.
 +
#Place the mixture on ice for 30 minutes. Do not mix.
 +
#Heat shock at exactly 42°C for exactly 30 seconds. Do not mix.
 +
#Place on ice for 5 minutes. Do not mix.
 +
#Pipette 950 µl of room temperature SOC into the mixture.
 +
#Place at 37°C for 60 minutes. Shake vigorously (250 rpm) or rotate.
 +
#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.
 +
 
==Solutions needed==
==Solutions needed==

Revision as of 06:36, 28 September 2011

Contents

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.

Solutions needed

For C2987H: Thaw a tube of NEB 5-alpha Competent E. coli cells on ice for 10 minutes. 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. 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. Place the mixture on ice for 30 minutes. Do not mix. Heat shock at exactly 42°C for exactly 30 seconds. Do not mix. Place on ice for 5 minutes. Do not mix. Pipette 950 µl of room temperature SOC into the mixture. Place at 37°C for 60 minutes. Shake vigorously (250 rpm) or rotate. 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.