Re: PCR Hint #1 No final 4 deg C step

Here is the first of what I hope will be a series of short hints for people doing PCR.

1. Don't set the final thermocycling step to 4 deg C.

This is something that I see all the time in people's PCR protocols - they set the final cycle step to 4 deg C. This is especially common with reactions performed overnight. This is not only unnecessary as the PCR product will not degrade if left at room temp for a few hours (after all any nucleases present will have been denatured at the 95 deg C step), but it is very hard on the PCR machines peltier unit. The peltier unit is usually the first thing to fail (and is very expensive to repair) so anything you can do to minimise its stress is a good idea. So instead of setting the final step to 4 C set it to 25 C. Nothing bad will happen to your PCR reactions if left at 25C and the PCR machine won't need to work keeping the block cold.

Daniel Tillett
Nucleics Support



Edited 2 time(s). Last edit at 12/21/2006 11:27AM by nucleics.

Interesting. I never thought about it before. We just had one of our pcr machines die and maybe this is what caused it. I think I will bring this up at our next lab meeting.

Yes cooling is one of the hardest things for the PCR peltier unit to do. If you you can one additional tip is to place the PCR machine is a well cooled room with good ventilation to help make the cooling step less of a strain.

In this light I once used to run an air cooled capillary PCR machine in a 4˚C cold room - it really helped speed up the thermocycling stage.

Daniel Tillett
Nucleics Support

Do you think that leaving RT-PCR products overnight (12-16h) at room temperature is too long? Will this cause the RT-PCR products to degrade? The RT-PCR product is still in the commercial reaction buffer (Mg, dNTPs, stabilizers), together with the enzymes, primers, water (and RNA).

There shouldn't be any difference between a RT-PCR product and a normal PCR product in regards degradation at room temp.

Daniel Tillett
Nucleics Support