Gibson Assembly

Gibson Assembly

赵晋平

Revised by Jinping Zhao

Jinpingzhao@https://www.wendangku.net/doc/1017116138.html,

1)Prepare 5× ISO Buffer:

Store at -20 ℃(200 reactions).

2)Prepare Gibson assembly master mix:

Store at -20 ℃in 5 μl aliquots (1 reaction).

*Optimized for 20-150 bp sequence homology overlaps.

3)Set up assembly reaction mixture:

Linearized vector backbone >100 ng; Each additional assembly piece ≧backbone molar Gibson assembly master mix (keep on ice until use) 5 μl ddH20 up to 10 μl.

4)Incubate the assembly reaction at 50 ℃for 60 minutes, then hold on 4℃or ice.

5)Transform 5 μl/1 μl of the assembly reaction into 100 μl of competent E. coli

Mach1-1 ultra-competent cell/ Top10 electro-competent cell. The expected efficiency is ~100/5 μl for Mach1-1 and ~1000/1 μl for Top10). The positive ratio is up to 90% for each method based on sequencing.