1) Prepare 6 ml of 5X ISO Buffer in a 15 ml falcon tube as follows:
3 ml 1 M Tris-HCl pH 7.5
+ 150µl 2 M MgCl2
+ 240 µl 100 mM dNTP mix (25 mM each: dGTP, dCTP, dATP, dTTP)
+ 300µl 1 M DTT
+ 1.5 g PEG-8000
+ 300µl 100 mM NAD
+ dH20 to 6 ml
Store at -20 oC in 320 µl aliquots.
2) Prepare 0.5 ml of Gibson Cloning master mix as follows:
320 µl 5X ISO Buffer
+ 0.64 µl 10 U/µl T5 exonuclease*
+ 20 µl 2 U/µl Phusion polymerase
+ 160 µl 40 U/µl Taq ligase
DO NOT ADD WATER
Store at -20 oC in 5 µl aliquots.This is 3.2 X, This mix allows you to add sample and water to 16 µl
This is optimized for 20-150 bp sequence homology overlaps
3) Thaw a 5 µl aliquot of the Gibson assembly master mix, and keep on ice until use.
4) Measure the DNA concentration (ng/µl) of each assembly piece.
5) Add 100 ng of the linearized vector backbone and equimolar amounts of the other assembly pieces in 11µl (e.g., elute DNA from spin column with 11 µl water after gel purification) to the thawed 5µl master mix in a 16 µl total volume assembly reaction mixture as follows:
linearized vector backbone (100 ng) + each additional assembly piece (to equimolar with backbone)==11 µl
+ 5 µl Gibson Cloning assembly master mix
6) Incubate the assembly reaction at 50 oC for 15 ~ 60 minutes, and then place on ice.