PCR trouble: about the annealing temperature - (Jul/20/2007 )

Hi, everyone. This is the first time I use this forum

The problem which i encountered these two weeks was the PCR. I want to amplify a region of a plasmid of about 2.6kb long. The 5' and 3' primers have high Tm (82 and 73 degree celcius). So i tried the following PCR conditions at my first trial:

Taq polymerase
Pre-Heating:
94 1min

30 cycles:
94 30s
62 30s
72 3min

Incubation:
72 10min
4 infinite

After the trial, a sharp 2.6kb band was shown in agarose gel electrophoresis, so i thought the conditions were suitable
However, when I want to reproduce the same result, i cannot amplify the region by using the same conditions. My supervisor told me to try other annealing temperatures: 50, 55, 57, 60, but all did not work. What could be the cause? Why I can't reproduce the result?
(All reagents, including the polymearse(Taq), are working. I used them to amplify other genes and all amplification are successful)
Actually i am thinking using even higher annealing temperature, say 65, 67...

Sounds to me that you are having problem with your DNA template.... Just check your DNA template first. Run a gel to check the quality of your DNA.

Others that I can think of is degradation of your dNTP.

With 50 as your annealing temperature, I am sure you will get at least unspecific bands.

Why not try gradient PCR? Easier that way to check your annealing temperature.

how about ditching that nasty Taq and get a proper high fidelity and high processivity polymerase, like KODhifi or Vent. Taq doesn't amplify well template over 1kb. While KODhifi will easily amplify your 2.6kb fragment.

I have a question though, why are your primer's Tm so high? Is the segment of the primer that binds to the template that long? Only the section of the primer that actually binds to the template is used to calculate the tm.

In addition to other suggestions, I would suggest getting out new aliquots of all components including primers for the PCR and trying it out with the same conditions as before.

Good Luck !!!

Maybe you can try a combined annealing and extension step at 72 degree, and 3 min 30 seconds? I would have tried an annealing temperature that is more matching with the high Tm of the primers.

Have you changed anything from the time when you got a product? New cDNA or something?