hi there,

I'm trying to PCR 1kb product using very long oligos. the For oligo is 118bp (18 anneal on the template and 100 are the tail) and a 69bp Rev (19 anneal on the template and 50 are the tail)

the template is a PCR fragment that I gel purified and is concentrated around 30ng/ul.

I'm trying to use the phusion taq from neb following the protocol but I got some of the PCR I need, but not all of them (the template is always the same, the only thing that changes in the different oligos is the tails)

so I tried gradient PCR, and I got some of the pcr amplicon that I failed to get using the normal PCR but not all of them.

I tried touch down pcr with very similar results as the normal Phusion taq protocol.

I also tried TAIL-pcr and I got some of the missing amplicon, just are quite dirty with extra bands.

I cant change the sequence of this oligos since I'm targeting a specific genomic locus.

any help with a pcr protocol/taq that can solve the problem is highly appreciated!