Is a 4°C Holding Temperature for a PCR Reaction Necessary?

PCR thermal cyclerPCRmax Alpha Cycler

By Michael Steinert, Product Manager, Biosciences, Cole-Parmer

Determining what’s best for your experiment vs. your thermal cycler

As a research scientist in my past life, I have been in the position as many others, where I simply left a completed plate of PCR reaction in a thermal cycler overnight—not at a holding temperature of 4°C or 10°C, but at room temperature. My immediate thought was that the reaction was ruined and that successful genotyping was impossible. However, to my surprise, the gel electrophoresis run with this “ruined” reaction provided the necessary separation and band concentration I needed to genotype my mice.

As it turns out, DNA is extremely stable even at room temperature for a short duration, and some studies suggest it can remain stable for days. This is why many in the scientific community use a holding temperature of 10°C instead of 4°C and why many newer thermal cyclers do not even allow for 4°C hold. There a few reasons for this, and the main argument is the stability of a completed PCR reaction allows for the high integrity of the reaction at 10°C while also putting significantly less strain on your thermal block.

Having the block at a low temperature such as 4°C can actually cause more condensation than a higher holding temperature. This condensation can actually cause damage to the block overtime, significantly reducing the lifespan of the machine. Not to mention, by simply setting your temperature that low, you are already requiring more work from your equipment. A 10°C hold will cause less wear and tear on your machine and lead to a piece of PCR equipment that runs without issues for years to come. However, for long term storage, it is best to store your completed reaction at -20°C to preserve the sample.

Of course, if you have been using a 4°C hold for much of your scientific life, I can see why you would be hesitant to give the 10°C hold a try. If you have enough sample, and time to compare, I would suggest seeing if the 10°C vs. 4°C hold shows any difference in yield or band visibility. If it doesn’t, you may have saved years of life for your thermal cycler, and saved thousands of dollars in the long run.

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References:

https://www.colorado.edu/ecenter/sites/default/files/attached-files/seracare_stability_of_genomic_dna_at_various_storage_conditions_isber2009.pdf

Jennifer Joiner, Molecular Staging Inc. New Technology Increases the Availability of High Quality DNA for Genetic Testing. CSR News, 2002 2.John G. Baust, Strategies for the Storage of DNA Biopreservation and Biobanking 6:251–252 (2008) 3.Lasken RS, Egholm M. Whole genome amplification: abundant supplies of DNA from precious samples or clinical specimens. Trends Biotechnol. 2003;21:531–53

John G. Baust, Strategies for the Storage of DNA Biopreservation and Biobanking 6:251–252 (2008)

Lasken RS, Egholm M. Whole genome amplification: abundant supplies of DNA from precious samples or clinical specimens. Trends Biotechnol. 2003;21:531–535.

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