|Line 69:||Line 69:|
Revision as of 09:33, 14 October 2013
Gibson Chew Back and Anneal Assembly (Gibson CBA) is a quick and easy method to construct plasmids without using restriction enzymes. In this method, DNA fragments to be assembled are PCR amplified with 40 bp of overlap to the adjacent sequence (only one DNA fragment per interface needs to have an overlap extension into the adjacent fragment). These fragments are then mixed in a single pot with a single strand exonuclease to generate sticky ends and allowed to anneal before being repaired by a polymerase and a ligase. In theory, many fragments can be assembled simultaneously (5, 4 inserts + backbone, have been documented in the initial report) into a single product of up to ~100 kb+. The method has several thermocycled variants of higher efficiency but the isothermal protocol offers almost comparable yields with greater simplicity.
- Master Mix
- Store in -20C Freezer
5x Isothermal Reaction Mix
- 3 ml 1 M Tris-Hcl (pH 7.5)
- 300 μL 1 M MgCl2
- 60 μL 100 mM dGTP
- 60 μL 100 mM dATP
- 60 μL 100 mM dTTP
- 60 μL 100 mM dCTP
- 300 μL 1 M DTT
- 1.5 g PEG-8000
- 300 μL 100 mM NAD
- 6 ml Total
Assembly Master Mix
- 320 μL 5X Isothermal Master Mix
- 0.64 μL 10 U/μL T5 exonuclease
- 20 μL 2 U/μL Phusion DNA Pol
- 0.16 μL 40 000 U/μL Taq DNA Ligase
- 1.2 ml Total
RFC57 Recipe 5x isothermal reaction buffer:
- 25% PEG-8000
- 500 mM Tris-HCl pH 7.5
- 50 mM MgCl2,
- 50mM DTT
- 5mM NAD
- 1mM each of the four dNTPs
1.33x Gibson Master Mix:
- Taq ligase (40u/ul): 50 ul
- 5x isothermal buffer: 100 ul
- T5 exonuclease (1u/ul): 2 ul
- Phusion polymerase (2u/ul): 6.25 ul
- Nuclease-free water: 216.75 ul
Gibson Method, aka VLIC, aka one pot isothermal assembly
Original protocol from: this paper, doi:10.1038/nmeth.1318
- PCR up your fragment of choice and gel purify
- Thaw a 15 μl assembly mixture aliquot and keep on ice until ready to be used.
- Add 5 μl of DNA to be assembled to the master mixture. The DNA should be in equimolar amounts. Use 10-100 ng of each ~6 kb DNA fragment. For larger DNA segments, increasingly proportionate amounts of DNA should be added (e.g. 250 ng of each 150 kb DNA segment).
- Incubate at 50 °C for 15 to 60 min (60 min is optimal).
- Transform as usual
- Ideally you have an overlap of 40 bp
- Misc: - rxn product is salty: can be a problem for electrocompetent cells. Rob Egbert dilutes the competent cells when transforming with electroporation. Not so much a problem when using chemically competent cells. - Electrocompetence is ~ 1000x more effective so is tempting.
- When preparing the isothermal reaction mix, add the PEG slowly to liquid. If added too quickly it will form a plug which will make mixing difficult (KS)
- The initial paper suggests that 10 - 100 ng of total DNA be used for assemblies. I've gone as high as 170 ng without any ill effects. (KS)
- Have successfully used for a two way and three way ligation (KS)
- There is a potential for mutations at the DNA boundaries which has yet to be quantified. Paper suggests 1 every 50 assemblies or so. Of the two initial assemblies I made, one had a missense mutation so sequence to verify interfaces or leave spacers (~ 50 bp or so) at the interfaces to 'absorb' these errors (KS)
- I have used PCRs as is (with PCR cleanup only) and gel extracted DNA in my assemblies. PCR cleanup gives more colonies (more DNA, better quality (no agarose/QG contamination)) but also has more false positives (PCR template plasmid). False positives may be alleviated by DpnI treatment if gel extraction is not used but I haven't tested this yet (KS).
- I once inadvertently designed my primers with 20 bp homology and a 20 bp spacer from the adjacent fragment and still got accurate plasmids. Possible to use less overlap if desired (KS)
- Guide to Gibson Assembly
- Gibson DG, Young L, Chuang RY, Venter JC, Hutchison CA 3rd, and Smith HO. . pmid:19363495.
Isothermal assembly method
- Gibson DG, Benders GA, Andrews-Pfannkoch C, Denisova EA, Baden-Tillson H, Zaveri J, Stockwell TB, Brownley A, Thomas DW, Algire MA, Merryman C, Young L, Noskov VN, Glass JI, Venter JC, Hutchison CA 3rd, and Smith HO. . pmid:18218864.
Two step thermocycled assembly variant