Some question about pad sequence


I’m trying to design a couple of sets of primers for 16S V1-V2(forward AGAGTTTGATCMTGGCTCAG and reverse ACTCCTACGGGAGGCAGC), Could you give me some concrete methods about how to make a suitable pad sequence and link sequence ? or about the principle of pad sequence and link sequence?

I am really grateful if anyone can provide some suggestions! Thanks very much in advance!


First, I would strongly discourage using anything other than the V4 region (see

Second, the linker sequence is a 2 nt sequence that is anti-complimentary to any other sequence in the database and the pad is a ~6 nt sequence that increases the Tm of the sequencing primer to between 60 and 65C. You can see the details about primer design and sequencing on the wet lab SOP: