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Problems with DNA gel extraction can be a real show-stopper since this is such a routinely used procedure. But, even if you are having no particular problems, it’s always nice to try and pick up some information that might improve your technique just that little bit.
Probably for these very reasons, Suzanne’s article 10 Tips for better DNA Gel extraction proved very popular. It seems like many of us are keen to get all the tips we can on this procedure. Well, if it’s tips you are looking for, we are always happy to oblige.
By scouring the recesses of my brain, colleagues and the internet I have squeezed out 5 more tips on DNA gel extraction. Maybe one of them will make the difference for you.
1. Turn the tube around. If you use Qiagen’s gel extraction kit, you may have noticed that after each spin, a small droplet tends to form inside the column at the side that was to the outside of the centrifuge rotor due to the shape of the column. It’s probably not too important during most of the procedure, but it’s worth bearing in mind at the elution step. At elution, the droplet is formed from residual PE buffer. Eluting with the droplet present will cause it to end up in your final sample, causing a bit of ethanol contamination.
When I heard the solution to this problem, it was one of those “why didn’t I think of that?” moments. Just do the 2 minute drying step after the PE wash in two steps. One minute with the column’s hinge to the outside of the rotor (drop forms on the outside), one minute with the hinge to the inside (drop is forced through the column). Brilliant!
2. Think about the elution volume. If you are worried about a low yield from your extraction, the natural tendency is to use a low elution volume. Beware of this. Using a low elution volume will increase the concentration of any contaminants that come off the column, as well as your DNA. This can be a problem for ligations and other sensitive applications so it is sometimes worth taking the lower concentration in return for a cleaner product. This solved some ligation problems I was having recently.
If you really want to go for the maximum concentration after gel extraction, it could be worth considering Zymo’s DNA gel extraction kit, as this allows elution with only 6 microlitres.
3. Wash, wash, wash. It is easy to underestimate the problems that residual agarose or salt can cause in some downstream applications. To avoid this, I always do an extra wash with the extraction buffer (e.g. QG in Qiagen’s kit) to remove excess agarose. And I always do an extra wash with the wash buffer and allow the wash buffer to stand on the column for two minutes prior to centrifugation to remove excess salts. These steps are so quick and easy so it’s worth doing them routinely if they can save you problems downstream.
Also, going by the “crap in, crap out” doctrine, another good approach is to minimise the amount of agarose you put onto the column in the first place. Trimming the gel slice as much as possible is always a good idea of course, but you can also reduce the amount of agarose you use in the gel. If you get good enough separation, why not use a 0.6% gel instead of 0.8 or 1%? It might help.
4. Use a gene catcher. Cutting bands (especially multiple bands) out of a gel can be tricky, and we all know that it’s a good idea to minimise the amount of UV exposure your band receives.
The people at The Gel Company have come up with a nice simple idea that addresses this problem. Their GeneCatcher disposable gel excision tips fit onto the end of a standard 1mL pipettor and have ends shaped like a standard gel band. Just push the tip into the agarose and your band will be forced into the tip. You can then eject the tip (containing your band) and go onto the next one.
At 25 US cents each they are a bit expensive, but could be worth it if they improve your results.
5. Guanosine – Sunblock for DNA. Instead of worrying about the amount of time that your DNA is exposed to UV you could always use sunblock. In a gem of an article (BioTechniques Vol 21, No 5: pp 898 – free registration required) researchers from the University of Heidelberg showed that 1mM guanosine added to agarose gels completely protected restriction-digested DNA from a 45 second exposure to 312nm UV, while in the absence of guanosine, this level exposure reduced ligation efficiencies by 2-3 fold. They also showed that guanosine did not affect downstream in vitro transcription or ligation.
So those are my DNA gel extraction tips. Do you have any you want to share?