(by customer from newsnet.com)
Thu Oct 11 06:06:35 EST 2007
> 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?
1. Unbalanced Tm and primer lengths doesn't help. Primers should end in one
or more G or C's.
2. Make sure the genomic DNA is clean.
3. I hope your using controls to ensure everything that should work is
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