Janet B. Matsen:Guide to Gibson Assembly
Back to Janet
- 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
- Electroporation is usually used to provide higher yield.
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
- 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
- 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
- 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
- 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.