I have never worked with hygromicin primers, but what I can conclude from your
history is perhaps that you are using too much gDNA in your PCR. Have you
tried different gDNA concentrations?, I mean, dil 1/10, 1/100, 1/1000 and
1/10000? Generally you should not see the gDNA you are using as template.
When looking to T annealing (Ta), my advice is that you should look for the
best conditions in your lab with the termocycler you are actually using to
make the PCR. According to the Tm you calculated (with the formula), the Ta
should be from 3 to 5 degrees lower than the lowest Tm calculated (i.e. for
your primers: Tm FWD = 70 and Tm REV = 66, you should start assaying Ta 60) I
don´t know how does it work with hygromicin primers, but that is the general
Hope it serves
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?