Janet B. Matsen:Guide to Gibson Assembly

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** Transform
** Transform
*** Electroporation is usually used to provide higher yield.  
*** Electroporation is usually used to provide higher yield.  
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==Procedure==
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=== Make a plasmid map of your design ===
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* This is key.  You will want it for primer design, checking your primers, assessing sequencing reactions, etc.  I use [http://biologylabs.utah.edu/jorgensen/wayned/ape/ APE], open-source software.  See my [[APE use page]]
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=== Design primers ===
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*
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=== Generate PCR fragments ===
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=== Purify PCR fragments ===
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=== Gibson assembly reaction ===
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== Transformation ===
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== Sequencing ===
== Examples ==
== Examples ==
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* Dpn1
* Dpn1
* RFP for backbone: don't screen red colonies!
* RFP for backbone: don't screen red colonies!
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== Making your own Gibson mix ==
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* Recipe
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* Tips:
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** Balancing the ratio of T5 & Phusion is mportant given the mechanism.  The exonuclease is so concentrated relative to the desired concentration in the mix that it should be diluted 10X before use.

Revision as of 21:34, 9 October 2012

Back to Janet

Contents

Intro

  • What is it?
  • How does it differ from other cloning?
  • When should I use it?
  • Steps (concise)
    • Design oligos to yield 40 - 100 bp overlapping linear DNA segments
    • Purify (usually gel) the PCR products (or digest)
    • Use Gibson Assembly Mix
    • Transform
      • Electroporation is usually used to provide higher yield.

Procedure

Make a plasmid map of your design

  • This is key. You will want it for primer design, checking your primers, assessing sequencing reactions, etc. I use APE, open-source software. See my APE use page

Design primers

Generate PCR fragments

Purify PCR fragments

Gibson assembly reaction

Transformation =

Sequencing =

Examples

  • you can chose where the seam is if you use longer oligos
  • Dpn1
  • RFP for backbone: don't screen red colonies!

Making your own Gibson mix

  • Recipe
  • Tips:
    • Balancing the ratio of T5 & Phusion is mportant given the mechanism. The exonuclease is so concentrated relative to the desired concentration in the mix that it should be diluted 10X before use.
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