by Karmella Haynes, 2013
Principle: The familiar "BioBrick cloning" enzymes (i.e., EcoRI, NotI, XbaI, SpeI, PstI) are Type II restriction enzymes, which cut the sequences that they specifically bind to. The Type IIS Assembly method method uses a Type IIS restriction enzyme, which binds at a specific sequence and cuts at a non-specific location exactly five base pairs away. As a result, the enzyme cleaves away its own binding site and leaves behind the most useful feature of assembly, sticky overhangs. When designed properly, Type IIS sites can be used to perform seamless assembly of parts. As an added convenience, this protocol allows cutting and ligation to occur in a single tube, as a single reaction. Thus, gel purification steps can be eliminated.
This protocol uses the Type IIS restriction enzyme BsmBI (CGTCTCnnnnn/).
Part Preparation - PCR
- Multiple parts can be assembled in one step.
- Parts and the destination vector should be amplified by PCR
1. First, map out the assembly. For instance: vector left - Part A - Part B - Part C - vector right. In this example, the vector, Part A, Part B, and Part C will be PCR amplified. 2. Design a pair of primers for each part and the vector...
- Forward Primer: 5'-cacaccaCGTCTCa + first 15 bp of "vector right" (top strand)
- Reverse Primer: 5'-cacaccaCGTCTCa + last 15 bp of "vector left", reverse complement (bottom strand)
Part A Primers
- Forward Primer: 5'-cacaccaCGTCTCa + last 4 bp of "vector right" (top strand)
- Reverse Primer: 5'-cacaccaCGTCTCa + last 15 bp of "vector right", reverse complement (bottom strand)