Janet B. Matsen:Guide to Gibson Assembly

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Revision as of 21:34, 9 October 2012 by Janet B. Matsen (Talk | contribs)
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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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