3
$\begingroup$

I'm using the Q5 system and I'm PCRing a product that will have a final length of around 9500 bases. I have noticed that the product is there but very faint. The primer seems to be used up.

So my question is, can the dNTPs be rate limiting in a long reaction? That is, can all the dNTPs be used up in the first PCR cycles and maybe not be available for the later exponential rounds?

It seems you have about 1000 times the amount of dNTPs to the amount of primer (200 µM compared to 0.2 µM). So maybe...or is my basic chemistry off. Any precedence?

$\endgroup$
  • $\begingroup$ IIRC Q5 requires 0.5µM of each primer as mentioned in documentation. In any case, why not just add more dNTP if you suspect it to be the cause? $\endgroup$ – March Ho May 27 '15 at 5:23
  • $\begingroup$ I am doing that now. But like anything in Biology, I'm sure there are consequences of adding too much. $\endgroup$ – jwillis0720 May 27 '15 at 5:31
  • $\begingroup$ Add controls in if in doubt $\endgroup$ – Rover Eye May 27 '15 at 7:58
  • $\begingroup$ Yes it is possible. dNTPs can also get degraded at high temperatures. Therefore they are added in excess. You can add more and as long as you are not doing real-time PCR don't worry too much about smeary/multiple bands. You can always do a re-PCR. $\endgroup$ – WYSIWYG May 27 '15 at 10:09
  • 1
    $\begingroup$ Adding too much dNTPs can cause problems. They bind magnesium from the reaction buffer, so if you are going to increase the amount of dNTPs drastically, you will have to optimize the reaction again for magnesium. $\endgroup$ – Chris May 27 '15 at 10:14
3
$\begingroup$

200uM dNTPs in 50ul reaction mix could produce more than 10ug DNA if all dNTPs were consumed whatever the length of PCR products is. You could detect as low as 5ng DNA fragments by conventional electrophoresis using EtBr. You probably could see a clear band when you load more than 100ng of a DNA fragment. Therefore I am not worried much about the dNTP concentration.

The longer PCR products you amplify, the more difficult it is to amplify. Then you may not get enough efficiency to detect your PCR products. You might want to try lower annealing temp if you do not get any none-specific bands with/without some additives such as DMSO, betain, etc., or use more stronger enzymes. Also touch down PCR may be useful to get specific bands efficiently.

When you increase dNTP concentration, Mg++ effects may be muted because dNTP could chelate Mg++ as Chris mentioned in the comment column.

| improve this answer | |
$\endgroup$
  • $\begingroup$ What do you mean by "stronger enzyme"? Q5 is one of (if not the) best commercially available DNA polymerases. $\endgroup$ – March Ho May 28 '15 at 5:21
  • $\begingroup$ Oh, I am sorry, I missed that part... Of course, Q5 is one of the strongest. But, it does not mean that other enzymes are not useful. So still, you have a chance to get more PCR fragments by other enzymes such as primestar. $\endgroup$ – 243 May 28 '15 at 17:05

Your Answer

By clicking “Post Your Answer”, you agree to our terms of service, privacy policy and cookie policy

Not the answer you're looking for? Browse other questions tagged or ask your own question.