Are there issues with filling PCR tubes to capacity?

Question

I'm planning to scale up a PCR reaction, and I'm wondering if filling the PCR tubes to the maximum volume of 200 ul would be a problem. It would mean a lot less pipetting as I would only need 1/4 of the tubes.

The typical protocols I've seen always use 50 ul for a PCR reaction, I'm wondering if there are any issues with larger volumes, e.g. differences in heat transfer, that could cause issues if I scale up the volume in one tube.

Is scaling up the volume problematic? Any specific aspects I should consider?

Answer 1

Too long for a comment: I would say it depends on the PCR. I have personally used maximum volumes of 50ul, if I needed to prepare higher volumes, I made one mix which subsequently got distributed to more tubes with 50ul max.

The problem with big volumes is to get a fast and homogeneous heating and cooling. If the outside of the tube is faster, the reaction there starts already while this may not be the case in the middle. This can lead to uneven denaturation and amplification of your template. The later is especially problematic when you amplify short sequences. There the amplification time might already be over and the PCR program is moving on before the template is finished, which will result in less of your full-length product and the enrichment of shorter versions.

There is another problem that might occur in big reactions: Due to gradients in reactivity because of temperature gradients I think it is possible that reagents (dNTPs, Mg-ions) can form gradients which further inhibit a good yield.

Finally I tested one thing on one of our PCR machines: The PCR tube is set into the heating/cooling block. It is not completely set into the block but stick out partly, so if you fill it up to maximum capacity, the upper 75-100ul (approximately) would be outside the heating block and we have a thermal problem. The reaction mix there would only heat up due convection.

Answer 2

(Also too long for a comment.) This question reminded me about an anecdote told by Arthur Kornberg concerning the approach to scaling up that was adopted by Reiji Okazaki (discoverer of Okazaki fragments).

Here is a quotation taken from For the Love of Enzymes: The Odyssey of a Biochemist by Arthur Kornberg  

Reiji’s research style is not readily gleaned from his publications, but it is vivid in my memory. One example I call the Okazaki maneuver. In purifying an enyme, he used a heating step: 10 milliliters of the enzyme solution was held in a test tube at 70°C for five minutes; then the coagulated impurities were removed by centrifugation. When he came to the point of scaling up the procedure to several liters, he simply repeated the original heating procedure several hundred times. I was embarrassed to have to report such an unsophisticated procedure in our publication. But then I realized that he was able to complete this step in a few hours and saw no point in wasting precious time and material in learning how to do the heating in a large beaker or flask. Later, when another of my students purified an enzyme with a heating step and had to scale up his procedure 2,000-fold from 3 milliliters to 6 liters, I advised the Okazaki maneuver: he added 3 milliliters to each of 200 test tubes to carry out the heating and centrifugation step, repeating the procedure nine times. Another student who tried heating a large volume of this enzyme in a single step lost it in a thick coagulum.