PSA: Your thermal cycler is NOT a refrigerator (Don’t hold below ambient!)

Published: June 10th, 2019   Last Modified: August 27th, 2019

One of the most precious machines in the molecular biology lab is the thermal cycler, often called the PCR machine. They dutifully cycle between the temperatures you set, amplify your DNA and never complain. We’ve got quite a herd ourselves and we love them all dearly. We’ve got Rowan, Cycler #2, Chad, River, Ol’ Red and Greybeard.

Unfortunately, these machines are often not treated with the respect of a 20,000$+ piece of gear and are instead used as a glorified mini-fridge. What do I mean by that? After your PCR is finished, do YOU set it to hold at 4C? Do you let it hold 4C overnight? Do you let it hold at 4C on a Friday and only turn off the machine Monday? Do you compromise and let it hold at 10C or 16C? Stop! Just stop!

There’s NO advantage to holding your PCR reaction at anything below ambient for any length of time! Let me try to convince you so you can SIGNIFICANTLY extend the lifetime of your machine.

This message is brought to you by the People for the Ethical Treatment of Thermal Cyclers (PETTC)

Thermal cyclers, how do they work? If you’ve read any advertisements for these machines you will have seen the term peltier module thrown around. Without getting into the chemistry and physics behind them, they’re sandwiches with ceramic plates as the bread and semiconductor materials as the peanut butter and jelly. You apply a current into one of these modules and one side of the module gets hot, the other gets cold. What does one look like?

Doesn’t look like much, but attach an aluminum plate filled with holes on one end, a heatsink/fan on the other, some control circuitry and voila, you have a thermal cycler! A high quality thermal cycler by Eppendorf can have 6-12 of these little rascals, usually individually characterized for that specific machine.

When treated with care, a high quality peltier module will last 50,000-100,000 hours (or even longer!). That’s 5-10 years if used all day, every day, around the clock. What kills them? Like us humans, time. Also, the act of thermal cycling itself. There’s not too much we can do about the thermal cycling, that’s its job. But we can certainly decrease the amount of miles put on the machine by not holding the machine after your program is done. Less stress on the peltier elements, less stress on the fans and on the power supply.

So you ask me, what do you want me to do Pipette Jockey??? Let my precious PCR tubes coast to ambient after the program is done? YES! Not only is your PCR reaction perfectly stable at room temperature for a few hours, it’s stable for over a week! Let me show you!

To demonstrate, I did a PCR for pCambia0305 in two sections, one approximatly 6.5 Kb and the other about 3 Kb. PCR was done with Q5 polymerase with GC buffer and 0.5 ng of plasmid. After the PCR was done, one set of tubes were put inside my desk at room temperature, out of the light. Another set was placed deep into the back of the fridge, to emulate parking your PCR machine at 4C. The last set I froze at -20C, which is the ideal scenario besides running your PCR on a gel right after it’s done. I ran all the samples on a 1.5% agarose gel a WEEK later, to simulate a graduate student forgetting about their PCR samples/going on vacation/finding themselves etc.

See any difference worth using up the lifetime of your thermal cycler on? Doesn’t look like it. Any sort of nuclease or contaminant will be annihilated after being cycled 30-40 times from 50-95C!

So please, adjust your PCR programs to finish after the final extension and let it coast the room temperature. Your PI will thank you since they won’t have to drop 20,000$ on a new machine prematurely, and your lab mates will thank you for keeping the machine alive for their experiments!

!!! Caveat emptor !!!
If you purposely have nucleases added into your reaction for your specific application, then you may have a legitimate reason to hold at 4C. Cycling may not destroy high concentrations of nucleases.

P.S. The above fragments will be re-constituted with Gibson Assembly, need a few days for some pictures. I want to show that after a week at room temperature, PCR fragments not only look good on a gel but are competent for cloning.