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 |
600 μl | 0.5 M MgCl2 (MgCl2 does NOT want to stay in solution!) |
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 480 μl aliquots.
2) Prepare 1.2 ml of 2x Gibson assembly master mix as follows:
Volume | Ingredient |
|---|---|
480 μl | 5X ISO Buffer |
0.96μl | 10 U/μl T5 exonuclease* |
30 μl | 2 U/μl Phusion polymerase |
240 μl | 40 U/μl Taq ligase |
449 μl | dH20 |
1.2 ml | Total Volume |
Store at -20C in 100 ul aliquots.
when you start a new aliquot, make 20 ul sub-aliquots in PCR tubes.
*This is optimized for 20-150 bp sequence homology overlaps
3) Thaw a 20 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 50 ng of the linearized vector backbone and appropriate amounts* of the other
assembly pieces to dH2O so that final volume is 5.0 ul.
*equimolar if fragment >1kb, otherwise 5-fold molar excess of fragment over backbone
Set up negative control in parallel: backbone + dH2O, no insert(s)
Add 5 μl master mix in a 10 μl total volume assembly reaction mixture as follows:
Volume | Ingredient |
|---|---|
| linearized vector backbone (50 ng) |
| each additional assembly piece (or none, for negative control) |
5 μl | Gibson assembly master mix |
| dH20 to 10 uL Total Volume |
10 uL | Total Volume |
6) Mix by pipetting. Incubate the assembly reaction at 50 C for 60 minutes, and then place on ice.
7) Transform 5 μl of the assembly reaction into 50 μl of competent E. coli and/or run a diagnostic agarose gel to check for successful assembly.