To those who have worked with hygromycin primers for genomic DNA:
I am trying to do PCR amplification of my genomic DNA (WT and mutant
lines). So far, I haven't had any luck in getting the Hyg primers to
hybridize to the DNA templates. I have observed various annealing
temperatures and wondering if these are the annealing temperatures
that are often used in hybridization of these primers.
5' AGC TGC GCC GAT GGT TTC TAC AA 3'
Tm = 61.70 deg Cel
5' ATC GCC TCG CTC CAG TCA ATG 3'
Tm = 58.84 deg Cel
For the PCR reactions I have used annealing temperatures ranging from
58 to 61 deg Cel. But none of these have shown PCR products. Should I
use a higher annealing temperature? When I manually calculate the
primer Tm using formula :
Tm = 4 (G+C) + 2(A+T), I get
Tm = 70 deg Cel forward primer and Tm = 66 deg Cel reverse primer -
both which are much higher than listed on the tubes.
I can see there is DNA after I run the gel, however no hybridzation
occurs. So, DNA is there. Are there any suggestions on making these
Hyg primers hybridize to my DNA templates?