Question

How would you resolve two DNA fragments of similar size as two separate bands on an agarose gel?

Answer 1

In order to resolve two DNA fragments of similar size as two separate bands on the agarose gel the following can be performed based upon the size of the DNA fragments that are needed to be separated:

If the length of DNA fragments are larger say greater than 8kb, we can use a lesser agarose gel concentration of about 0.7% and run the gel for a longer duration (about 2.5 hrs) for the fragments to be obtained as separate bands.

If the length of DNA fragments are smaller say about 3-6kb, we can use a greater agarose gel concentration of about 1.0% and run the gel for a longer duration for the fragments to be obtained as separate bands.

Means smaller the DNA fragments higher the agarose gel concentration must be used and vice-versa.

Apart from above two methods if the sequence of fragments in known we can use restriction enzyme to cut one of the fragments into two further fragments and then perform the usual gel electrophoresis in order to separate fragments. But, this can be only used in case of requirement of only one fragment which is not cut using restriction enzyme.

In case of DNA fragments having length greater than 15kb which will travel similarly through gel, we can use Pulse Field Gel Electrophoresis (PFGE) which applies switching of direction of electric field periodically in three directions:

One in the direction of central axis of gel and other two at an angle of 60 degrees on either side of gel. This process takes a longer duration in comparison to usual gel electrophoresis.