Hi everyone,
I was running a PCR and, unfortunately, the thermocycler died around the 7th cycle. It felt like a waste to simply throw away the PCR, so I put it on another thermocycler and lowered the cycle number to 27 (it is usually 34). Has anyone had experience with changing thermocyclers during PCR and what were your results? Should I even use this PCR for downstream steps or should I just re-do it entirely?
The thermocycler settings are:
1) 94C, 3 min
2) 94C, 30 sec
3) 55C, 30 sec
4) 72C, 45 sec
5) Go back to Step 2, 34x (again, I lowered this to 27 when I changed thermocyclers)
6) 72C, 20 min
7) Hold on 4C
Thank you.
After 7 cycles you have amplified the product 2exp7 or about 128 times as much as you started with so when you restart the pcr the amplimer is common compared with the non specific sequences so I would expect the pcr to work well and would only have concerns if the product is being cloned but I would expect the product to sequence well if that is the next step
It sounds like you've outlined a clear plan for managing the thermocycler interruption. Just to summarize your approach:
This approach should help you continue with minimal disruption to the PCR. Good luck with your amplification, and I hope the samples come out well!
If you finished the PCR you could let us know if it worked. In principle, there should be no problem to continue with the rest of the program on a second cycler. Remember, in the early days of PCR there were no thermostable polymerases, so they had to add the enzyme after every cycle of the reaction, so interrupting the program for a few minutes should still give you enough product.
Let us know how it worked for you. I hope you could solve it. In my opinion the PCR will work just fine even if you continue with another cycler with similar conditions. Ultimately you wish to amply the sequence right! So if your primers and all are already standardised accordingly I think the sequence surely will get amplified. The number of cycles is less means less number of amplication. The product still gets amplified.