1) Prepare 6 ml of 5X ISO Buffer in a 15 ml falcon tube as follows:
Volume | Ingredient |
|---|---|
3 ml | 1 M Tris-HCl pH 7.5 |
150 μl | 2 M MgCl2 |
240 μl | 100 mM dNTP mix (25 mM each: dGTP, dCTP, dATP, dTTP) |
300 μl | 1 M DTT |
1.5 g | PEG-8000 |
300 μl | 100 mM NAD |
| dH20 to 6 ml Total Volume |
6 ml | Total Volume |
Store at -20 C in 320 μl aliquots.
2) Prepare 1.2 ml of Gibson assembly master mix as follows:
Volume | Ingredient |
|---|---|
320 μl | 5X ISO Buffer |
0.64μl | 10 U/μl T5 exonuclease* |
20 μl | 2 U/μl Phusion polymerase |
160 μl | 40 U/μl Taq ligase |
| dH20 to 1.2 mL Total Volume |
1.2 ml | Total Volume |
Store at -20 C in 15 μl aliquots.
*This is optimized for 20-150 bp sequence homology overlaps
3) Thaw a 15 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 to the thawed 15 μl master mix in a 20 μl total volume assembly reaction mixture as follows:
Volume | Ingredient |
|---|---|
| linearized vector backbone (100 ng) |
| each additional assembly piece (to equimolar with backbone) |
15 μl | Gibson assembly master mix |
| dH20 to 20 uL Total Volume |
20 uL | Total Volume |
6) Incubate the assembly reaction at 50 C for 60 minutes, and then place on ice.
7) Transform 5 μl of the assembly reaction into 100 μl of competent E. coli and/or run a diagnostic agarose gel to check for successful assembly.