How to design PCR Primers for cloning into C-terminal fusion vectors pTXB, pTYB (pTYB 1,2,3 and 4), or pCYB vectors?
The following examples demonstrate how to design primers used for PCR reactions to generate a target gene fragment with (1) an NdeI, NcoI, or BspHI site at its 5´ end, (2) an XhoI (pTYB2) or SapI (or BspQI) site at its 3´ end, or (3) blunt-end(s). The sequence corresponding to the sequence of the putative target gene is in lower case. Restriction enzyme recognition sequences are underlined. The addition of extra bases (NNN NNN) 5´ to the restriction site are required for efficient cleavage by the corresponding enzyme. Although the length of such a sequence may vary according to a specific enzyme, 6 bases are generally recommended. Please note the SapI isoschizomer BspQI (NEB #R0712) can be used instead of SapI.
