My target sequence is either approximately 200 or 260 base pairs, and I am seeing the longer product being unexpectedly favored in my PCR reaction. Should I shorten my extension time from 30 seconds to something lower (or get rid of it completely) to get more representative results?
Required elongation time depends on the DNA polymerase and template DNA you're using. While you may try to shorten the extension time, it is likely you have another binding site for one of your primers and playing with annealing temperature (rising it) may prove more helpful. It might help even more to set up a temperature gradient +- 5 degrees around annealing temperature of your primers.
I agree with Pavel that increasing the annealing temperature or adding DMSO should be tried first. If you want to try decreasing the extension time that would be fine but do not forget that the polymerase is active at all temperatures so the annealing time could be shortened as well . There are enzymes on the market using only 1 second for annealing and extension times. Thermal cyclers take a while to change temperatures and the reaction happens at a range of temperatures so if your cycler increases temperature at about 3degrees a second then annealing and extension still takes place over quite a few seconds
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