Janet B. Matsen:Guide to Gibson Assembly: Difference between revisions
From OpenWetWare
Jump to navigationJump to search
| Line 20: | Line 20: | ||
** 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. | ** 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. | ||
* Use cheap primers | * Use cheap primers | ||
** 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 *** | ** 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: we pay ~$9 for a 60 bp primer but ~ $34 for a 61 bp primer. 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 === | === Generate PCR fragments === | ||
Revision as of 18:59, 9 October 2012
Back to Janet
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 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.
