Prather:Gibson CBA

Gibson Chew Back And Anneal Assembly: One Step Isothermal
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.

Reagents
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


 * balance  ddH2O


 * 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
 * 860 μL 	ddH2O
 * 1.2 ml	Total

Aliquoted reaction and master mixes are stable at -20°C and can withstand several freeze thaw cycles.

Protocol

 * 1) PCR vector and insert(s) ensuring that at least 40 bp homology exists between adjacent fragments
 * 2) Thaw assembly master mix and keep on ice until ready to be used
 * 3) Mix 15 ul of assembly mixture with 5 ul total of cleaned PCR product (PCR Cleanup Kit or Gel Extraction) keeping DNA inserts in equimolar amounts
 * 4) Incubate at 50 °C for 15-60 min (60 min optimal).
 * 5) Transform cells with no more than 1 ul of assembly mixture.

Comments

 * 1) 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)
 * 2) 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)
 * 3) Have successfully used for a two way and three way ligation (KS)
 * 4) 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)
 * 5) 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).
 * 6)  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)