Janet B. Matsen:Guide to Gibson Assembly

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Revision as of 22:06, 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

  • The primers should confer 20-100 bp of homology between to adjacent overlapping segments. 40 - 100 bp is ideal; substantially shorter or longer will give you lower yields.
  • The annealing portion of the primer should have Tm between 62oC and 65oC as calculated by this Finnzymes website
    • This formula is applicable to Phusion DNApolymerase, the DNA polymerase used to form the DNA you will assemble.
  • Use cheap primers
    • If ordering with IDT, primers should be 60 bp if you are encoding homology. The price per base pair jumps when you add the 61st base pair: we pay ~$9 for a 60 bp primer but ~ $34 for a 61 bp primer. Using less than 60 bp reduces the length of the homolgy between adjacent DNA pieces in the assembly. Note: there are cases when you use standard size (18-22 bp) primers as is discussed in this page. *** DISCUSS ***
  • Check primers for cross dimers with Finnzyme's multiple primer analyzer. If the annealing temperature of the primer dimer(s) is low, this will probably not be a problem during PCR.
  • Make sure the reverse primer is reverse complemented!

Generate PCR fragments

Purify PCR fragments

Gibson assembly reaction

Transformation

Sequencing

Examples

  • Break up backbone if it is large (> 4kb??)
    • Only need 2 short primers to break it up: the homology is free.
  • you can chose where the seam is if you use longer oligos
  • 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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