I have used gradient PCR at four temperatures 54, 60, 61 and 62 to standardize annealing temperature for my polymorphism. at three temperatures i.e. 54, 61 nd 62, good intensity bands are being shown, but when i used simple endpoint PCR at 61 annealing temperature, my wells are empty.
Did you run t least one sample of dna that worked on the gradient PCR?
Is the pcr cycle exactly the same as with the gradient pcr.
Is anyone else using the end point pcr machine and getting good results.
It is possible that all the new dna samples are impure and contain pcr inhibitors but if you have not changed your reaction volume and cycle parameters then I suspect that either the denturing temperature is a problem or that the plastic of the tubes/plates used on the end point pcr have changed the pcr profile so that the correct temperature is not being reached.
I would try modifying the pcr profile so that the reaction mix has a longer time at each temperature so increase the denaturing time and the annealing time by 50% and see what you get. It may be that the ramp speed of the gardient machine is slower than the end point machine and giving the pcr longer at high and low temperatures allows the reaction mix more time to equilibrate at the correct temperature.
You can also decrease the annealing temperature by 1c for the same reason while you are troubleshooting
- Badges
- Science method
- Similar topics
- Biological Science
- Ecology