Hi guys,
I'm going to qualitatively test my DNA polymerase with PCR. But something cross my mind, will my DNA polymerase denature since it is a protein? And I assume it should be quite heat sensitive. The optimum activity of my DNA polymerase is 65 degree celsius.
Is there any way that I can test it qualitatively? Thanks!
Usually you will use a heat stable Polymerase for PCR (e.g. Taq Poylmerase, "Taq's optimum temperature for activity is 75–80°C, with a half-life of greater than 2 hours at 92.5°C, 40 minutes at 95°C and 9 minutes at 97.5°C" (from Wikipedia)). Other, non heat stable, polymerases will of course denature.
You can test the functionality of your polymerase by running PCR with different temperatures, and check if you get you desired product.
I agree with mr Hansen, you can try using various temperatures in PCR. Normally Polymerase used in PCR are stable in high temperature though. However don't use temperature exceeding your primers Tm to avoid it unbind from DNA before elongation process starts.
It depends on your DNA sample (length, GC content,...). Usually 95°C are used for denaturation of DNA.
If the DNA strands are not separated at the temperature you choose, your PCR will not work.
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