Janet B. Matsen:Guide to Gibson Assembly

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* Make sure the reverse primer is reverse complemented!
* Make sure the reverse primer is reverse complemented!
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=== Double Check your Design ==
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=== Double Check your Design ===
* Blast your primers and templates with [http://blast.ncbi.nlm.nih.gov/Blast.cgi?PAGE=Nucleotides&PROGRAM=blastn&BLAST_PROGRAMS=megaBlast&PAGE_TYPE=BlastSearch&SHOW_DEFAULTS=on&BLAST_SPEC=blast2seq&QUERY=&SUBJECTS= blastn] and make sure they only anneal where you expect.
* Blast your primers and templates with [http://blast.ncbi.nlm.nih.gov/Blast.cgi?PAGE=Nucleotides&PROGRAM=blastn&BLAST_PROGRAMS=megaBlast&PAGE_TYPE=BlastSearch&SHOW_DEFAULTS=on&BLAST_SPEC=blast2seq&QUERY=&SUBJECTS= blastn] and make sure they only anneal where you expect.
* Blast the APE files for the expected PCR products against each other to make sure they have the correct amount of overlap.  Make sure there is not extensive homology in other places.
* Blast the APE files for the expected PCR products against each other to make sure they have the correct amount of overlap.  Make sure there is not extensive homology in other places.

Revision as of 22:02, 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: 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!

Double Check your Design

  • Blast your primers and templates with blastn and make sure they only anneal where you expect.
  • Blast the APE files for the expected PCR products against each other to make sure they have the correct amount of overlap. Make sure there is not extensive homology in other places.

Generate PCR fragments

  • I run each PCR at a few annealing temps and DMSO concentrations. See my sample spreadsheet.
  • [http://www.neb.com/nebecomm/products/productr0176.asp Dpn1 can be added after the PCR is complete to degrade the template DNA. This will reduce the number of background colonies when you transform.
  • Run a few uL of each PCR product on a gel to identify rxn conditions that yield a lot of product.

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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