Reaction volume

Q: Does the PCR reaction volume (negatively) influence the outcome?

A: No, especially since the introduction of the small, thin walled, 0.2 ml plastic vials fitting the 96 well metal blocks of the thermocyclers.

A number of observations are worth mentioning:

if using older model thermocyclers (without a heated lid), to run small volume PCR reactions, mineral oil is necessary to cover the reaction mixture. Use of oil increases the volume of liquid in the vial and thus can influence somewhat the outcome. Besides, older thermocyclers require the use of larger plastic vials, with thicker walls, that fit less well in the metal blocks and thus may increase the likelihood of variation in PCr outcome.

small, thin-walled plastic vials designed for the 96 well metal blocks are ideal for running small-volume PCR reactions. Due to the heated lid of the thermocyclers, there are no mineral oil requirements. When tested, reactions yielded similar results, whether the reaction volume was 100, 25 or 5 microliters.

It is important to mention, that small volume PCRs may be very beneficial when using small amounts of DNA template. In general, at a constant amount of template DNA, the yield of PCR product per microliter reaction is higher when the reaction volume is 5 µL compared to 100 µL. This may allow visualisation of the PCR products, sometimes invisible when larger reaction volumes are used.