Janet B. Matsen:Guide to Gibson Assembly

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Revision as of 09:35, 9 October 2012 by Janet B. Matsen (Talk | contribs)
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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.


Examples

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