Gibson Assembly

• NEBuilder HiFi DNA Assembly Reaction Protocol

• Gibson Assembly Master Mix

• NEB Protocol

• Gibson, D.G. et.al (2009). Nature Methods. 343-345.

• Gibson, D.G. et al. (2010). Nature Methods. 901-903.

• Barnes, W.M. (1994). Proc. Natl. Acad. Sci.. 91, 2216-2220.

CBAR (Gibson Assembly)

This protocol is deprecated and is no longer used. Use the HiFi Master Mix protocol.

CBAR Mix Recipe

Source

1. Prepare 5X ISO buffer. Six ml of this buffer can be prepared by combining the following:

2. 3 ml of 1 M Tris-HCl pH 7.5

3. 150 μl of 2 M MgCl2

4. 60 μl of 100 mM dGTP

5. 60 μl of 100 mM dATP

6. 60 μl of 100 mM dTTP

7. 60 μl of 100 mM dCTP

8. 300 μl of 1 M DTT

9. 1.5 g PEG-8000

10. 300 μl of 100 mM NAD

11. Add water to 6 ml

12. Aliquot 100 μl and store at -20 °C

13. Prepare an assembly master mixture. This can be prepared by combining the following:

14. 320 μl 5X ISO buffer

15. 0.64 μl of 10 U/ μl T5 exo

16. 20 μl of 2 U/μl Phusion pol

17. 160 μl of 40 U/μl Taq lig

18. 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.

1. Thaw a 15 μl assembly mixture aliquot and keep on ice until ready to be used.

2. 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).

3. Incubate at 50 °C for 15 to 60 min (60 min is optimal).