Separation of smaller DNA fragments and larger fragments without using gel electrophoresis?

Question

For an upcoming experiment I would have two kinds of DNA fragments in a sample after a particular reaction step: First there would be fragments of a length of approx. 170 bps and then there would be Fragments of a length of approx. 270 bps or more.

I would like to maintain all fragments with a length of 270 bps or more and throw out all fragments with a length of 170 bps.

Of course, I could run everything on a gel and collect just those bands above 170 bps. However, I am wondering whether there is something more elegant to reach that aim. For instance, we could use a filter that collects just those fragments with a size of 200 or 250 bps or more and those fragments with a smaller size flow through the filter. Is such a thing possible and if so, how? Or would you suggest something else?

Many thanks for your support in advance!

Answer 1

One option is to use SPRI beads, like those sold by Ampure (Ampure XP beads) to separate DNA fragments by size. By adjusting the ratio of beads to sample, you can selectively bind fragments of certain lengths by excluding those of other lengths, as seen in the following image:

From this image, you might try using a beads-to-sample ratio of 0.7 to exclude fragments below 200bp, or a ratio between 0.7 and 0.6 to exclude only those fragments below somewhere between 200 and 250bp. The advantage to beads over a gel is the time it takes. Size selection by beads takes only around 10 minutes, versus maybe 30 minutes or more for a gel (including the extraction).

As with any molecular biology procedure, it's important to test it and make sure that it works for you. For instance, it might be a good idea to take aliquots of your samples and compare the lengths and yields that you get from a range of bead concentrations and/or to compare the yields that you get from using gel vs (in this case) beads.

Answer 2

In my mind, gel separation (using a fairly high percentage gel to separate the bands as much as possible) and manual excision is by far the best option. The problem with molecular weight cutoff filters is that the value they give (5000 Da, for example) is an average pore size, it is not exact. Therefore, any attempt to separate 270 bp from 170 bp oligos will end up with some of the shorter construct ending up in the final product, and some of the longer sequence in the flow-through. The chances of finding an off-the-shelf product that exactly meets your size requirements are also low. Only use these types of kits if you're separating a 300 bp PCR product from unused primers, for example.

You could do some sort of column chromatography using a size-exclusion matrix, but that's essentially the same thing as using a gel. The only advantage is it can be scaled, so if you have large amounts of PCR product to separate (let's say 5 or 10 ml), it would be easier to run a single column than to load that many lanes in a gel.