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I'm going to be running some Taqman assays on cDNA generated from RNA collected from various samples, and will end up running a bunch of plates (right now the setup is 1/2 a plate per sample, including controls etc.). Since our thermocycler can only handle one 96-well plate at a time, it has been suggested that I make up the mixes all together, aliquot them into the plates, then store the plates at 4°C (well wrapped in plastic or Parafilm) until ready to run. I would imagine that the plates would be in the fridge for just one day, at max overnight.

My question is about the polymerase - can I add it ahead of time, and just take the plate out of the fridge and pop it in the thermocycler, or should I add it just before use? I've been kind of out of the DNA/RNA field for over 10 years, and I seem to remember from before that polymerases could have residual activity even at 4°C. Is this still the case, or have they improved to only be active at 60+°C or whatever your temperature is?

Alternatively, is there another ingredient I should leave out until just before amplification? cDNA? Probes? Something else? Also, does the storage time make sense? Is overnight okay, or should I run them all the same day? Is over the weekend okay? I don't want this question to extend into a too-broad "How do I do qPCR?" question, but any tips you have would be very helpful. As far as I'm aware, all the reagents are from Life Technologies/Applied Biosystems.

The reason I ask is because it seems to me that it would be much more accurate and reproducible to prepare the master mix with polymerase already included, instead of adding 1 μL of polymerase to each well just before running the assay.

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  • $\begingroup$ I don't see why you can't just freeze all the samples, and then prepare the master mix and run the PCR in one single go. Purified cDNA is far more stable than PCR mixes. $\endgroup$ – March Ho May 9 '15 at 20:16
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    $\begingroup$ I stored several pre-mixed (with all the components, the polymerase included) plates in a -20˚C freezer for up to 1 month and still worked beautifully. Was using Taqman reagents. Would opt for the freezer rather than the fridge. Just take out your ready-to-amplify plates a bit ahead of time to allow them thawing. $\endgroup$ – cagliari2005 May 9 '15 at 22:28
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    $\begingroup$ @MarchHo the cDNA has been prepared ahead of time and frozen. On the day of the qPCR, I prepare the reaction using 2 dye-labeled probes (gene of interest and internal standard), PCR master mix, template, and H2O. All this is aliquoted into 96-well PCR plates and stored at 4C. When it's time to run the next plate, it's taken out of the fridge, polymerase is added, the plate is put in the thermocycler, and run. What I'm wondering is whether I can add the polymerase when I'm setting up the plates, then just take the next plate out of the fridge, put it in the thermocycler, and run. $\endgroup$ – MattDMo May 9 '15 at 23:07
  • $\begingroup$ I would suggest freezing it at -20 rather than at 4. You will still have some activity at 4, and its best to avoid that. $\endgroup$ – Rover Eye May 10 '15 at 9:11
  • $\begingroup$ It is much easier and better to set up the reaction again. After a run, the thermocycler (for Real-Time such as light-cycler) should generally be allowed some rest (1h). In this duration you can prepare your second plate. You can aliquot cDNAs in PCR tubes (or strip tubes) with your probes and keep them at -20. You can add taqman master mix with appropriate volume of water before the reaction and load the PCR plate just before running the PCR. $\endgroup$ – WYSIWYG May 11 '15 at 5:44
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Not sure if this question is still valid, but it was marked as unanswered and frequently viewed, so here's a go :)

These days most polymerases (certainly for Real Time PCR) are so-called Hot Start enzymes (ask your manufacturer, because they save you a whole lot of trouble!). They are modified with a protein that prevents them from working until activated at 95 deg. Celsius. Be aware though that not all hot start enzymes require the same activation time at 95 deg. Celsius, and some enzymes lose activity when activated for 15 min. at 95*C instead of 2 min.

In my experience storing plates at 4 degrees (or room temperature) is not a problem at all, and most manufacturers advertise that the PCR mixes (including primers, probes and DNA) are stable at room temperature for 24 hours. You do however need to protect the mixtures from (sun)light because the probe might be damaged when exposed for longer times.

Personally, I would not recommend storing real-time PCR plates in the freezer, because condensate will most-likely form when you thaw your plates. This can influence the PCR, as real-time PCR uses light for the analysis of your samples.

If you want to save yourself some pipetting, but can't prepare plates in advance, you can prepare a primer-and-probe mix, which you can freeze in batches. In our lab, we keep a stock of primer-and-probe mix for every assay. Maybe this also works when you already add water and PCR mastermix, but I have never tried. I wonder how other people do this, so please leave a reply.

Hope this might help some people.

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