Debunking the 4° C Polymerase Chain Reaction (PCR) Myth
Myth:PCR product needs to be refrigerated if left overnight in the thermal cycler.
Truth 1: DNA will be stable for days, even weeks, inside a PCR tube.
Truth 2: Extended cold holds will reduce your PCR machine’s lifetime.
Many scientists and other polymerase chain reaction (PCR) users believe samples need to be refrigerated immediately after thermal cycling finishes. This results in users waiting for the reactions to finish so that they can place the tubes in a fridge or freezer, or programming thermal cyclers with an infinite 4˚ C hold.
Waiting for a reaction to finish can be inconvenient, though, and leaving a thermal cycler cooling overnight can severely reduce its useful life.
We decided to explore this issue in greater detail . . .
Do samples need to be refrigerated immediately after PCR?
The answer is NO. If amplification is successful, the DNA in the tubes will be stable for a long time. We did a simple experiment to confirm this. We amplified an 800-base-pair fragment. The amplicon corresponded to a DNA control for a miniPCR® Learning Lab. For 12 replicate tubes, we ran 25 PCR cycles in a miniPCR® Thermal Cycler. Following cycling, we left the 12 tubes at room temperature, freezing replicates once a day for 12 consecutive days.
Results analyzed by DNA gel electrophoresis are shown below. The verdict is clear: PCR product will not degrade when left at room temperature overnight, or even for 12 days.
Replicates from a PCR amplification were left for varying numbers of days at room temperature. Scenario: 15µl/well were loaded on a 2% TBE agarose gel stained with a green nucleic acid stain visualized on a blue LED transilluminator. The first lane is the 100 bp DNA ladder from New England Biolabs. Ambient temperature was ~22˚ C.
In the same figure, there is another example of the stability of DNA: ladders used as molecular weight markers in gel electrophoresis, such as the 100 bp ladder, can also be left out at room temperature for months without noticeable DNA degradation. (The ladder in the figure had been at ambient temperature for over 90 days before this run.) DNA ladders are often composed of fragments amplified by PCR.
Reasons to not leave your thermal cycler cooling all night
Most thermal cyclers in the market heat and cool using Peltier or thermoelectric cooling elements. While this is a great technology, thermoelectric elements are notorious for failing under heavy use and are extremely fragile. They are especially prone to breakage during transportation. Peltier elements have a finite lifespan, and every moment a thermocycler is left on counts toward that span.
Consistently leaving a PCR machine cooling overnight can reduce its lifetime by tenfold*, so turn off the machine the next time you’re doing PCR. If you use a miniPCR® Thermal Cycler, you don’t need to worry about this as the unit does not use Peltier elements and automatically shuts off after cycling.
Be aware of condensation with cold hold
Cold holds, or holds at 4 to 10 degrees Celsius, can cause air moisture to condense on the metal block that holds PCR samples. After a long cold hold, you may notice that the block appears “sweaty” or beaded with moisture. This condensation can damage the block over the long run. If water finds its way to the underside of the block, it may drip into the electrical components, causing electrical malfunction.
Corollary: DNA has evolved to be a very boring (stable) molecule
After all, the DNA double helix evolved to preserve our genetic information. Paleontologists amplify ancient DNA from bones deposited 50,000 years ago, and forensic scientists routinely collect DNA evidence from messy crime scenes. If we can study DNA from samples that have been in the environment for hundreds and even thousands of years, it is to be expected that our PCR bands will survive a night at room temperature.
So give your thermal cycler a break and get a good night’s sleep knowing your samples are safe.
*Based on thermal cycling for 90 minutes and overnight cooling (15 hours).