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I have performed DNA precipitation using two methods on an amplified 194 bp PCR product:

  1. using a PEG method
  2. with just cold 80% ethanol

PEG worked much better for me.

Can the same methods be applied to qPCR products? If not, could someone propose a way of doing that? I am in the exploration stage, and I am trying to learn different experimental techniques.

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    $\begingroup$ You need to provide some more information: What is your PEG protocol? How big are the qPCR products you want to precipitate? How much yield do you expect (this can make a big difference in the methods you can apply)? $\endgroup$
    – Chris ♦
    Commented Nov 8, 2023 at 6:45
  • $\begingroup$ @Chris thanks for your reply. To answer your questions. 1) First stage, 1:1 Peg/ PCR Product volume and two washing stages with 75% ethanol. At the end of each stage, I performed spinning at 4oc / 15000rpm for 15 mins, disposing of the supernatant, and finally, letting the pellet dry (open tube lid for 15 mins at room temp). 2) The qpcr product is also 194bp. 3) The yieldiest, the better. I just want to practice. $\endgroup$
    – KGee
    Commented Nov 10, 2023 at 13:54

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I would say, in general the answer is yes, with caveats.

Firstly, remember that the optimal amplicon size for qPCR is small, so you can expect lower yields.

Secondly, what is the primer concentration that you use in qPCR? If the primer concentration that you found to be optimal for the purposes of quantification is low, then the amount of the final product will be that much lower, as at some point, qPCR will run out of primers to produce new amplicons.

Thirdly, if you’re using probe-based qPCR, remember that you might have non-specific amplification there that doesn’t show up on the amplification plots, because there is no probe hydrolysis when the non-specific product is amplified. If you’re using DNA-binding dye-based qPCR and your melt curve gives you a single peak, you can be relatively sure that you have just the one PCR product (although, theoretically, you could still have two that just happen to be of the exact same size).

A tip I can offer is that for the precipitation of tiny amounts of DNA (or RNA) it is a good idea to add some molecular biology grade glycogen (or glycoblue, which is glycogen stained blue to make the pellet more visible) – provided your downstream applications aren’t glycogen-sensitive. Although I only used glycogen in isopropanol/ethanol precipitations, I’m not sure whether / how it would interact with PEG.

Hope this helps.

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