hygromycin primers

SVemuri12 from gmail.com via methods%40net.bio.net (by SVemuri12 from gmail.com)
Sun Oct 7 17:18:53 EST 2007


Hello,

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.

Forward primer:
5' AGC TGC GCC GAT GGT TTC TAC AA 3'
Tm = 61.70 deg Cel

Reverse primer:
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?



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