- Prepare 5X ISO buffer. Six ml of this buffer can be prepared by combining the following:
3 ml of 1 M Tris-HCl pH 7.5
150 μl of 2 M MgCl2
60 μl of 100 mM dGTP
60 μl of 100 mM dATP
60 μl of 100 mM dTTP
60 μl of 100 mM dCTP
300 μl of 1 M DTT
1.5 g PEG-8000
300 μl of 100 mM NAD
Add water to 6 ml
Aliquot 100 μl and store at -20 °C
- Prepare an assembly master mixture. This can be prepared by combining the following:
320 μl 5X ISO buffer
0.64 μl of 10 U/ μl T5 exo
20 μl of 2 U/μl Phusion pol
160 μl of 40 U/μl Taq lig
Add water to 1.2 ml
Aliquot 15 μl and store at -20 °C. This assembly mixture can be stored at -20 °C for at least one year. The enzymes remain active following at least 10 freeze-thaw cycles.
This is ideal for the assembly of DNA molecules with 20-150 bp overlaps. For DNA molecules overlapping by larger than 150 bp, prepare the assembly mixture by using 3.2 μl of 10 U/ μl T5 exo.
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).
If cloning is desired, electroporate 1 μl of the assembly reaction into 30 μl electrocompetent E. coli.