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We ran PCR with a positive control and two lanes of sample. We used the same sample, but in one PCR mixture we used a total volume of 50μL (25μL Taq, 5μL forward&reverse primer each, 10μL sample, 5μL dH2O) while in the other we halved each volume to a total of 25μL. Pipetting was done by the same person.

The positive control and the 25μL total volume lane gave nice and clear bands, but the 50μL sample showed nothing. How could this be explained?

Edit: We only ran one PCR with all the samples simultaneously, so no second thermocycler or different temperatures or anything. Didn't even consider that option!

Edit 2: In case it's relevant, we were using this: http://www.chemheritage.org/discover/collections/collection-items/scientific-instruments/perkin-elmer-cetus-model-480-dna-thermal-cycler.aspx

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  • $\begingroup$ Could you attach a picture of the gel? Also, how many times was this experiment repeated? I have used different volumes many times with no difference in efficiency (but with Phusion polymerase) $\endgroup$
    – Gergana Vandova
    Feb 26, 2013 at 4:34

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The thermal cycler adjusts sample temperature based on the volume of the sample. So if you run samples of different volumes side by side, not all of them will cycle through the optimal temperatures of each step. If you were working with a "hot start" polymerase this is even more critical, as the Taq won't be able to amplify at all unless the sample is heated up to a specific temperature.

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  • $\begingroup$ But presumably, @Armatus used different thermocyclers and he entered the appropriate volume of the sample in the thermocycler options. $\endgroup$
    – Gergana Vandova
    Feb 26, 2013 at 6:16
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    $\begingroup$ @GerganaVandova I didn't presume either that they did or didn't. In both cases it's still a probable reason for this problem. I imagine an ideal set of answers as all probable reasons for a problem. Then anyone looking this up in the future could use the answers to troubleshoot. $\endgroup$
    – Drosophila
    Feb 26, 2013 at 11:52
  • $\begingroup$ Does this apply to very old models? How would the thermocycler test the volume in a tube or the temperature it reaches? We weren't using a hot start. $\endgroup$
    – Armatus
    Feb 26, 2013 at 14:56
  • $\begingroup$ Older models simply raise the block to the temperature and don't worry about controlling for getting the liquid in the tube to the indicated temperature. In this case, it is possible that the real temperatures were different depending on volume. $\endgroup$
    – Jack Aidley
    Feb 26, 2013 at 15:02
  • $\begingroup$ That sounds like the most likely problem; I think you should post that as an answer. $\endgroup$
    – Armatus
    Feb 26, 2013 at 17:40

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