Agarose Gel Electrophoresis

Biology
Tutor: None Selected Time limit: 1 Day

1. Which percentage of agarose is better in resolving high molecular weight DNA fragments, 0/7% or 2.0%

2. Define the charge of

A) Pole closest to wells 

B) Net Charge on DNA 

C) Pole furthest from the wells

3. Which fragment of DNA would you expect to find closer to the wells where the DNA was loaded a 100bp fragment or a 3500bp fragment? Explain 

Apr 23rd, 2015

1. A gel made of 0.7% is better for resolving DNA fragments of high molecular weight. A gel that has a lower percentage of agarose will have a larger pore size, so larger DNA fragments can migrate more readily than a gel that has a high percentage of agarose.  A 2.0% agarose gel would have a smaller pore size; small pores impede the movement of high molecular weight DNA fragments and are best for resolving low molecular weight fragments.

2.

A) The pole closest to the wells is negative.

B) DNA has a net negative charge (because of its phosphate backbone).

C) The pole furthest from the wells is positive. This positive pole pulls the negatively charged DNA through the agarose gel, while the negative pole repels the negatively charged DNA away from the well.

3. You would expect to find the 3500bp fragment closer to the loading wells than the 100bp fragment. DNA fragments of low molecular weight readily fit through the pores of an agarose gel, can move more quickly through the gel, and thus migrate further from the wells than do DNA fragments of a high molecular weight. In contrast, DNA fragments of high molecular weights are impeded from movement by the agarose matrix and available pores, and will move much more slowly through the gel. For any DNA sample, at the end of electrophoresis, DNA fragments will be separated by size such that the larger fragments are near the wells and the smaller fragments are at the opposite end (closer to the positive pole).

Apr 29th, 2015

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