We ran PCR with a positive control and two lanes of sample. We used the same sam

Question

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 50?L (25?L Taq, 5?L forward&reverse primer each, 10?L sample, 5?L dH2O) while in the other we halved each volume to a total of 25?L. Pipetting was done by the same person.

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

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!

Explanation / Answer

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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