PCR is carried out in a buffer that provides a suitable chemical environment for activity of DNA polymerase. The buffer Tris-HCl concentration (high/low) can interfere with the pcr reaction.

If the study protocol is not yet optimized regarding the Tris-HCl concentration in the PCR reaction, the best in-house way to know whether the concentration is inhibitory is to run control reactions/samples with various Tris-HCl concentrations (with all other components held constant). Such consideration is highly dependent on the specific context of your laboratory, reagents and other instruments.

Hi Aedrian,

I routinely rehydrate my primers in 0.1X TE buffer and use them at 1:10 in the final reaction ( 0.01 X TE, (EDTA at 0.01 mM). Which is slightly lower than you are using, however the low levels of TE present in the primers should not materially interfere with the PCR. The EDTA level is so low in comparison to the divalent cations in most PCR buffers (typically 1-3 mM) as to render it insignificant (perhaps a 1% difference in the amount of available cation). There is far more variability in divalent cation concentration due to pipetting than you will be in introducing by the low levels of EDTA you are adding. The final buffering capacity of typical PCR buffer (Typically 10mM, pH 8.3 at 25C) so about 5X the concentration of tris you will be adding for the primers. The 20% increase in tris concentration in the final reaction should not interfere. Depending on the pH of the tris you used( e.g. pH 7), may slightly lowers the reaction pH and marginally impact the processivity of the taq. As long as you are not trying to minimize the extension time, you should be fine. Effects of small variations in pH again should not materially impact the primer annealing.

I hope this was helpful.

Frank