Hello,
I am running PCR for genotyping. I didn't have problem till this Summer, when I couldn't see any band on the gel. I checked primers, Taq, etc. I bought new stocks etc. But still I can't get any result. The only thing I changed compared to before, is the Thermal Cycler machine, but everyone was telling me that it was impossible my bad results were due to a malfunctioning of the machine. I then went to check the manuals of the 2 machines and I noticed that the main difference is their ramp rate: 1 degree celsius for the machine I am using now vs 4 degrees for the previous machine. Can this affect my PCR cycles and, therefore, my genotyping results?
thank you
Hi,
Read this article
https://www.thermofisher.com/in/en/home/life-science/cloning/cloning-learning-center/invitrogen-school-of-molecular-biology/pcr-education/pcr-thermal-cyclers/pcr-thermal-cyclers-considerations.html#:~:text=Ramp%20rate%20of%20a%20thermal%20cycler%20and%20synthesis%20rate%20of,3%C2%B0C%2Fsec).
I found the same article, and it says "Since the ramp rate of a thermal cycler can affect PCR results", but in other pages it looks that the ramp rate only affects the global time, so just the speed and not the quality. Since I don't have access anymore to the previous machine, do you know if there is an ideal ramp rate for genotyping cycles?
I think that the ramp time will effect the efficiency of the pcr because the samples remain longer at active temperatures in the slow ramp cycles. In a faster ramp speed there is also more overshoot at set temperatures so if denaturing at 94 then if you approach 94 quicly then the block will probably reach 96 briefly so small samoles may reach 96 or 95 with better denaturation. Similarly if approaching an annealing temperature of 55 very quickly then the block may go to 53 and then a small sample may reach 53 or 54 and better/different annealing may occur which will make a difference in how the pcr works. Sample volume and plastic tube/plate wall thickness will also make a big difference in how a pcr works
Thanks Paul Rutland. I thought the same and also on the 1 degree machine it is stated that "Unfortunately, the heating and cooling is slower than some PCR machines available at only 1oC per second. This means that the cycles will take longer than many other instruments – although, of course, this is not intended to be a high-throughput machine. The cycler is not capable of running temperature gradients across the block as is the case with many other machines."
Now, how can I decide what is the best ramp rate in my case? is there an ideal range, let's say from 2 to 3, or fastest possible (in my case 5) is better in any case?
Paola Salmas Indeed, ramp rate affects the PCR product, specifically your primer annealing. Since you have standardised your protocol with old machine, use the same or again standardise your PCR program with the new machine.
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