For the moment we're using 4°C as the holding temperature for our more valuable/sensistive samples, and for more forgiving experiments like generating amplicons from plasmids were using 10°C to reduce wear and tear on the thermocycler. I'm wondering if the 10°C would cause any significant sample loss or degradation for more sensitive samples, or if others have noticed any significant thermocycler loss-of-function from using prolonged 4°C holding temperatures? (Such as for overnight runs).
If you can add temperature gradients to your thermocycling, you could try running the same reaction with two different hold temps and run some QA/QC your products after holding overnight.
That's a good idea. We do have a gradient-enabled thermocycler, and I have no shortage of my current plasmid I could play around with to test this out on it.
I personally hold all my PCRs at 15°C, a colleague of mine even does it at 20°C, i have never experienced a problem with my PCR products (at least not concerning the holding temperature ;) I would assume that there are not many nucleases which are able to withstand the harsh temperature conditions of a PCR program.
Hold at 20oC. Less wear and tear on the block which prolongs the life. I haven't noticed any significant loss of sample after holding at 20oC (even overnight). Besides, 30+ cycles up to 95oC, down to about 60oC then 72oC, would kill off any nuclease contaminants.
We usually hold at 7°C, and we havent get any problem. It have been working normally as in 4 or 10 °C, that i usually see other people does. I think 7°C is a medium level.
The companies said: for long life of the cyclers not less the 10°C. That works without any problem.
Had never noticed any difference between RT, 4°C or 10 °C hold temperatures. For avoiding wear on the block I usually avoid 4°C. So I would not worry about degradation except in some applications (e.g. if you use UTP and heat stable DNA-glycosylase in your reaction, and perhaps direct PCR with dirty samples).
We have used a holding temp of 20oC. Same as David and Ferdinand.
We hold at both 10°C and 15°C and they have no problem, although our thermalcycler's company recommendation is 15°C for longer machine life.
I usually hold at 7°C but at 10°C or more (15-20°C) overnight. We haven't got any problems.
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