Amount of liquid:
5 micro liters master mix
5 micro liters of patient sample
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$\begingroup$ Please clarify your specific problem or provide additional details to highlight exactly what you need. As it's currently written, it's hard to tell exactly what you're asking. $\endgroup$– Community BotCommented Mar 15, 2022 at 19:50
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$\begingroup$ Welcome to the site - can you please let us know what sort of PCR you are doing. Regular PCR with a gel as end-point, it might not matter too much (i.e. you just want a band), but for qPCR it will make all the difference. $\endgroup$– bob1Commented Mar 15, 2022 at 22:12
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$\begingroup$ If your pipets are calibrated and your technique is good, you shouldn't have issues with those volumes. Using a p10 instead of a p20 might reduce pipetting error a bit. Low-binding tips can help too, but marginally. If running reactions in duplicate or triplicate you can mix them all in one tube first, and dispense the 10 ul reactions out from that; just make them around 10% or so bigger than what you need to account for dead volume, and wet your tips before dispensing. $\endgroup$– MikeyCCommented Mar 16, 2022 at 14:24
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Pipetting PCR reactions should be done as precise as possible - since relatively small deviations can effect the reaction. Pipetting 4 instead of 5µl results in a 10% error which for example leads to a higher magnesium concentration which may effect the reaction efficiency.
I would try to use bigger volumes (25µl is pretty common for PCR reactions) to minimize such effects, but of course smaller reactions save material and costs. You could run a few tests to see how big the effect is for different reaction volumes and if the possible error is acceptable for you.
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$\begingroup$ Could you clarify more on what would be an acceptable range of deviation in that it wouldn't effect the reaction or provide me please with any reference I could view? For example if I had a total mixture of 20µl. $\endgroup$– NicoleCommented Mar 15, 2022 at 20:38
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$\begingroup$ @Nicole you can work out the error by yourself fairly easily - it's simply your pipetting volume error divided by the expected total volume (e.g if you had a 1 ul error in expected volume 20 ul - 1/20 = 0.05 = 5%) $\endgroup$– bob1Commented Mar 15, 2022 at 22:16
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