Janet B. Matsen:Guide to Gibson Assembly

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(Design primers)
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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]]  
* 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]]  
=== Design primers ===
=== Design primers ===
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*  
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* 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.
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* The annealing portion of the primer should have T<sub>m</sub> between 62<sup>o</sup>C and 65<sup>o</sup>C as calculated by this [http://www.finnzymes.fi/tm_determination.html Finnzymes website]
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** This formula is applicable to [http://www.google.com/url?sa=t&rct=j&q=&esrc=s&source=web&cd=1&ved=0CDEQFjAA&url=http%3A%2F%2Fwww.neb.com%2Fnebecomm%2Fproducts%2Fproductm0530.asp&ei=8NV0UL6-BaP1igLzsoGABQ&usg=AFQjCNEAXwNrh7KmjNNLXmdlaS1n74xitw&sig2=hnZPEtJmOMyRtqLdiSe_IQ Phusion DNApolymerase], the DNA polymerase used to form the DNA you will assemble.
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* Use cheap primers
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** If ordering with [http://www.idtdna.com/site IDT], primers should be 60 bp if you are encoding homology.  The price per base pair jumps when you add the 61st base pair.  Using less than 60 bp lessens the base pairs of homology between adjacent DNA pieces in the assembly.  There are cases when you use standard size (18-22 bp) primers as is discussed in this page.  *** DISCUSS ***
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=== Generate PCR fragments ===
=== Generate PCR fragments ===
=== Purify PCR fragments ===
=== Purify PCR fragments ===

Revision as of 21:58, 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

  • 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. Using less than 60 bp lessens the base pairs of homology between adjacent DNA pieces in the assembly. There are cases when you use standard size (18-22 bp) primers as is discussed in this page. *** DISCUSS ***

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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