Size Determination of DNA Restriction Fragments?

Question

Anyone know how to determine the fragment size in base pairs? Then determine the percentage error with the experiment? I'm stuck.

Answer 1

Mass-spectrometry (MS) is the only technique that gives true quantitative answers with extremely high certainty, but MS of DNA is uncommon and hard to do. A more typical approach would be to "run a gel" separate the different fragments based on size: you have a porous matrix, you place the mixture of DNA pieces on one end, you apply a voltage. Since the DNA is charged it migrates on the gel, but the bigger pieces are hindered by the matrix (the gel). You then run a "standard" side-by-side with your unknown mixture--something with a mixture of a few fragments on known size. Typically these "standard ladders" which are commercially available come in increments, e.g. 100 bp, 500 bp, 1000 bp, etc. So you will be able to tell the size is between those fragment lengths (e.g. that a band from your unknown mixture is between 100 and 500 bp) with very high certainty, but you do not know the exact length with any certainty.

Alternatively you can sequence DNA--this is very easy and cheap now-a-days, and has a very high-certainty. So after doing a gel you can cut out the different bands and have them sequenced individually. Both steps--the gel and the sequencing--are very routine, cheap, and reliable.