Small scale PCR:ing and the components.

[Dr. Jekyll]

I'm interested in small scale PCR, but I'm just a layman in this area. I got some questions regarding the components needed. I start with the dNTP.

 

dNTP-mixes (or the individual dATP, dCTP, dGTP, dTTP) are often sold in quantities of around 0.5ml with a concentration of around

10-100mM. As I got it, quite often you only would like to use 50ul with a concentration of around (say) 50-100uM per vial.

If I do large scale PCRs I would probably dilute and use up most of the dNTP-mix I bought. But if you're doing small scale PCRs with say just 1-2 vials at the time,

how is the dNTP handled/stored, since it is not wise to freeze/melt dNTP more than a couple of times? Is it possible to dilute the bought (say 0.5ml, 100mM) dNTP and store/freeze it in quantities of 0.05ml?

Seems as a pretty tiny amount to freeze, but is this how to do it?

 

Hopefully you get what I'm aiming at, and hopefully I've understood it correctly. That is, the bought dNTP needs to be diluted and stored in small quantities, since otherwise 90+% would go to waste when doing small scale PCR.

[Bluefunk]

Freeze-thawing dNTPs can be problematic. I generally freeze and thaw no more than a couple of times before discarding. Small aliquots are the best way. Diluting the NTPs is fine, we use TE buffer to dilute.

 

But basically, yes. the bought dNTP needs to be diluted and stored in small quantities,

[Dr. Jekyll]

Oki, thanks!

 

I have no intuition regarding the size of such small quantities, e.g., 0.05ml. Didn't even bother to make a simple calculation of it, since I figured it would be kinda like dipping a needle in a cup of water . But, it is much larger than I thought: [math](0.05\times 10^{-3})^{1/3}\approx 3.7\textrm{mm}^3[/math].

[CharonY]

For most intents and purposes diluting the nucleotides in ultrapure water (sterile and DNA/DNAse free, of course) is preferable, unless you are only going to use one specific condition for your PCR (the absence of additional buffer makes certain applications easier, but often it is of no concern). Besides aliquoting them, it also helps to thaw them on ice. Ow, and you are aware that you need pipettors to handle microlitre volumes?

[ecoli]

not to mention Taq polymerase and a thermocycler.

[CharonY]

Well, yeah (though any other thermo stable polymerase would also do). And while we are at it, buffer, Mg2+ or Ca2+ (depending on polymerase), primer, template, small cups, ultrapure water... hmm did I forget anything (well, disregarding stuff like betaine etc.).

 

 

But I mentioned that because there appeared to be some confusion regarding the volumes.