All of us know that it is safe to store PCR product at -20 degree. But recently, i have come across certain strange things which i need to clarify. Some regions (trnH-psbA) were amplified two weeks back and amplification was confirmed by running 1.2% agarose gel. These products were stored in -20 freezer. Later 2 days back, when i again run the same products in 1.2% gel, I could not see even a single band any of the lanes. What might have caused this?...
When I asked my colleague about this, he opined that storage at -20 might be the issue and advised me to store PCR product in 4 degree.
Is it true that pcr product will be denatured/degraded in -20?
Rahul, Yours is a strange problem. We have not experienced such an issue. some times back some of our PCR tubes were accidentally left on the work bench for almost a week, but the products were fine and gave good clones & sequences too. But in -20 degradation seems to be very unusual. I am not sure, but may be, the tubes got contaminated with DNases post-PCR, possibly during loading the gels ??
SD
Dear Rahul
- Most if the time UNPURIFIED PCR products are stable for months at a time at -20C. I keep them at -20C unpurified for that reason
- what is more freeze thawing has no adverse consequences; as implied by your colleague
- Occasionally however a product will become degraded
- This is due to DNAse contamination; either picked up during handling or present in situ and acting cumulatively over time. To reiterate we are talking unpurified PCR reactions
- For that reason storage of PCR amplicons in the fridge without purification is generally not recommended for more than 1 week
- With purification PCR reactions are stably indefinitely at -20C; unless contaminated during handling by DNAses
It is always advised to purify the amplified product (either from the mix immediately after PCR or from gel. In the case of purification immediately after PCR it is better not to use master mixes with stain for PCR reactions) and resuspend it with TE buffer, pH 8.0, then store at -20 C for long term storage. No issue..
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