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Experiments & Protocols

Experiments

Protocols

Golden Gate

BsaI is a restriction enzyme with unique type 2 properties. What this means is that a sequence of DNA can be designed to have a desired overhang on each side. The BsaI will cut itself out of the sequence, as shown in the picture below:

After cutting its recognition sequence out and leaving the desired overhang, a gRNA with matching sticky ends can be inserted into the space. In other words, the gRNA will completely take the place of the BsaI restriction site.

The BbsI Restriction site can be added via PCR in order to create custom matching sticky ends to string multiple gRNAs in a row. This is known as Golden Gate Assembly.

BbsI is then used to snip out the gRNA to prepare for combining with more gRNAs in Golden Gate Assembly.