Hey guys,

I am trying to PCR out 0 to -1k region of three genes. I used pfu-ultra and followed strictly the protocol. I adjusted everything to their proposed working concentration. On top of that, I performed touch-down PCR with annealing temp changing from 65 degree to 55 degree and proceeded to 25 extra cycles with annealing temp set to 55 degree.

Luckily, I got 2 out of 3 promoters PCRed out. However, for the third one I kept getting smear. Please see attached image.

The primers for the last one are:

TACACGCGTGGAATTGAACTCAGGACCTCTA melt temp 63degree

TATCCTCGAGAAGAACCATAATAATCGGGGTA melt temp is 59degree

GC contents are 48% and 41% respectively.

For template, I use 50ng 3T3 genomic DNA.

Thank you all in advance!

Xingchen