Golden Gate Assembly is a method for assembling multiple DNA fragments, relying on the activity of Type IIS endonucleases that cleave outside of non-palindromic recognition sites.
In this example, the destination vector is designed to include BsaI recognition sequences, in opposing directions, to allow for the assembly of the desired fragments. Using PCR, BsaI recognition sites are incorporated into the fragments in the proper orientation through primer design.
If a destination vector with BsaI flanking sequences is not available, BsaI sites can similarly be introduced into any plasmid sequence by PCR to make a linearized form of the destination vector. In either case, the PCR-amplified fragments are ready to be assembled into the destination vector in a single-tube reaction containing BsaI and T4 DNA Ligase.
Cleavage with BsaI exposes complementary sequences for fragment assembly. A final short 55-degree Celsius step at the end of the assembly reactions favors digestion, ensuring only linear DNA remains.
Note that the correctly assembled fragments no longer contain BsaI sites, and therefore only the plasmids with the correct insertion will remain intact and ready for the last step, which is transformation.
Insert fragments can also be precloned into plasmids flanked by BsaI sites. During assembly, inserts and vector will ligate to yield the final assembled product, which lacks BsaI sites. Again, only the plasmids with the correct insertion will remain intact and ready for transformation.
For a list of other type IIs restriction enzymes that can be used for Golden Gate Assembly or other applications, please visit www.neb.com/TypeIIsTable
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