Does it affect the results if I use the half of dNTP amount in a reaction that it says in my protocol? I usually use 4 microlitres per reaction and this time I did a mistake and I used 2.

The final cDNA will be used for qPCR relative cuantification,so I am not sure if that will affect my results.

  • $\begingroup$ It depends on several parameters like what is the concentration of dNTP you use, the length of the DNA amplified, the polymerase and other PCR parameters. I think the best way to know would be to check the quality of your result via agarose gel electrophoresis. $\endgroup$ – Dart Feld Nov 17 '16 at 17:00

You will probably be fine. Lower dNTP levels might reduce the amount of product you obtained since it is usually the primers or the dNTPs that run out near the end of the reaction. The only other effect I am aware of is that the reaction might be slightly less specific as dNTPs sequester magnesium which is a big factor in determining the stringency of the reaction.

You say how many microliters you used but it would be more useful if you mentioned the concentrations of the dNTPs and the desired final concentration.

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  • $\begingroup$ Please cite content that supports your answer, opinion based answers are bad. $\endgroup$ – Dart Feld Nov 17 '16 at 16:56

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