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
Generate PCR fragments
Purify PCR fragments
Gibson assembly reaction
- 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.