Running Gels Backwards to Select DNA Molecules Larger than a Minimum Size

Many procedures require size selection of DNA molecules for subsequent cloning or for other procedures. If the desired DNA fragments are of a known specific size, then one can easily cut the required band from a gel and extract the DNA; there are many effective methods to purify DNA from the small piece of agarose that contains the band. It is much less convenient if one needs to select molecules spanning a range of sizes, because they run to different positions in the gel, so that one would need to cut a large piece of agarose to obtain the full range of sizes desired. It is most difficult if one needs molecules larger than a given size; to obtain all such molecules, one would need to cut a piece of agarose extending from the movement limit for large DNA molecules down to a position corresponding to the minimum desired size. Methods that are effective in purifying DNA from small pieces of agarose are often cumbersome for large pieces; even worse, extraction from large pieces of agarose can result in significant amounts of impurities in the extracted DNA. We have used a simple, fast, and effective procedure to select DNA molecules larger than a given size and to gather them into a small piece of agarose for easy extraction. We run a low-melt agarose gel containing the DNA to be size-selected (along with ethidium bromide and size standards). When the gel has run far enough to accurately locate DNA molecules of the minimum desired size, we cut the gel at that boundary and discard the portion of the gel with molecules that are too small. We replace the running buffer and then run the remaining part of the gel backwards at the same voltage as in the initial run. At the time the gel is cut, the desired DNA molecules lie in an extended, although possibly very faint, smear extending from the cut toward the well. The position of each molecule in the smear depends on the molecule’s mobility, which is determined by its size. If the electric current is reversed, so the voltage drop per centimeter of gel is the same as before (but now points in the opposite direction), then the DNA molecules will move back toward the well. Each molecule will move backwards with the same velocity that had previously carried it forward, to its position within the smear when the gel was cut. In effect, the molecules reverse their paths. All of these reversed paths converge at the well, so the smear of DNA gradually compresses into a narrow band as the DNA approaches the well. We monitor the progress of the convergence with a hand-held UV light (models FB-UVM-80 or FB-UVLS-80; Fisher Scientific, Pittsburgh, PA, USA). Shortly before DNA begins to move across the well, we cut the small piece of agarose just in front of the well that contains the converged DNA molecules. We extract the DNA using standard methods; for DNA greater than 2 kb, we find efficient recovery using agarase digestion (Roche Molecular Biochemicals, Basel, Switzerland). The motion of DNA through a gel includes a random, diffusional component that makes a DNA band gradually thicken as it migrates. Running the DNA backwards does not undo this diffusional spread, so the thickness of the converged band, and the zone within the converged band that is occupied by DNA molecules of each size, reflects the total duration of the run and the distance that the DNA molecules have moved through the gel. As a result, the converged band is at least as thick as an ordinary band of DNA (containing same-sized molecules) that has migrated a comparable total distance through the gel. To minimize this thickness, one may prefer to cut the gel and reverse the migration direction after a shorter rather than longer run. In addition to saving time, this allows the converged band to be excised within a smaller piece of agarose. Without diffusion, optimal convergence would occur precisely at the well, but diffusion limits the minimum thickness of the converged band so all of the DNA does not enter the well at the same time. As a result, the DNA is easiest to excise just before the band enters the well. In practice, we use the same total voltage when running the gel forward and in reverse. The electrical resistance of the gel is higher than that of the liquid buffer, so removing a portion of the gel lowers the total resistance of the circuit and increases the voltage drop per centimeter of the remaining gel. This somewhat increases the velocity with Benchmarks