Protocol to retrieve unknown flanking DNA sequences using semi-site-specific PCR-based genome walking

Here, we describe a protocol based on semi -site -specific primer PCR (3SP-PCR) to access unknown flanking DNA sequences. We specify the guidelines for designing primers for 3SP-PCR. We also describe experimental procedures for the 3SP-PCR, along with PCR product purification and subsequent sequencing and analysis. For complete details on the use and execution of this protocol, please refer to Wei et al.1