Thread: Discuss how each of the following factors would affect the results of electrophoresis?

a. Voltage used.
b. Running time.
c. Amount of DNA used.
d. Reversal of polarity.

EASY 10 points, I just need be be reassured that my answer is correct.

1a higher voltage would cause you to achieve the 'result' faster
b. longer running time will cause the bands to travel further and further from the starting well
c. larger amount of DNA would make the resulting bands appear thicker (assuming you're using stain) and more visible
d. reversal of polarity will simply cause the bands to be formed on the other side of the wells.

Voltage applied affects the migration time of the samples. It can aso afffect the quality of the sample migration. If the samples are run at a high voltage they will run faster, and typically more smeary. The higher the voltage the hotter the gel will run. You need to be careful for certain applications with low-melting agarose. If run too high, you could melt your gel.

Running time ... as the gel runs longer, you typically can obtain better resolution (separation) of bands. Although this is not the only factor that affects the separation, it is worth pointing out that gel percentage plays a role, too. High gel percentage = better resolution of small bands (and vice versa.) Eventually, if left to run long enough, the smallest bands will run off the gel first.

Amount of DNA used. If you don't use enough DNA, you will not be able to see it on the gel upon staining. If you use too much DNA, it can clog the well, and be difficult to resolve. Keep in mind as the DNA runs, each band represents some % of the total amount of DNA loaded.

Reversal of polarity ... this would cause the DNA (or RNA or protein) to run in the opposite direction, ie backwards from the well, than you want.

For DNA, it should be run from negative to positive b/c DNA is negatively charged, so it will run toward the positive end. If you inadvertently switch polarity, your DNA will go backwards out of the well and into the buffer.