Two sets of primers are used to amplify the gene of interest:
- 15-bp homology regions to the vector, flanking the cloning site into the insert.
- Specific gene sequence. If the sequence has 5' overhang, the start of the 15 bp homology must begin from the 5' most extension to include the overhang; if the sequence has a 3' overhang, homology should begin where the DNA becomes double-stranded. Please refer to the manual for illustration of primer design.