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