Anyone who has worked in a molecular biology lab knows that DNA gel extraction can be surprisingly challenging. Why is this? Is it because of poor product yields, or maybe it’s because the gel extraction process uses harsh chemicals and conditions (e.g., chaotropic salts, ethidium bromide, ethanol, heat) that will damage or denature DNA and potentially decrease cloning success.
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.
In this article, we share some more tips to help you maximize your yields of high-quality DNA from the gel extraction process. By scouring the recesses of my brain, colleagues and the internet I have squeezed out these tips on DNA gel extraction. Maybe one of them will make the difference for you.
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Top 15 DNA Gel Extraction Tips
1. Trim the Gel Slice as Much as Possible
Get rid of all excess gel, including the gel in front of or behind your DNA band. Most people cut out a square around the gel but don’t think to stand the excised piece up and trim the gel away from the front and back. If you pour a thick gel, there will be a lot more gel to remove. The more you can remove from your DNA band, the higher your yield will be.
2. Minimize Exposure to UV Light
The UV light causes DNA damage that can impact the clonability of the DNA. Cut your gel slice quickly. If you have multiple bands to trim, work with one band at a time under UV. Don’t leave your entire gel sitting under the UV light while you cut one slice at a time. Otherwise, you risk that the DNA exposed to UV for the longest time will be nicked to shreds. Alternatively, use a visible range stain such as methylene blue or crystal violet. You can read more about this step in our article on preparing vectors for cloning.
3. Remove All Traces of Phenol Using A “Home Brew” Method
If you are using phenol to purify the DNA from agarose, bear in mind that phenol traces will not be removed by ethanol precipitation and will inhibit subsequent ligation reactions. You can evaporate traces of phenol from the aqueous phase by warming the supernatant to 65°C for 5 minutes. Then, let it cool back down to room temperature over 10-20 minutes before precipitating to ensure that you obtain double-stranded DNA (remember that double-stranded DNA separates a high temperatures).
4. Change to a New Brand or Bottle of Agarose
Sometimes, agarose actually causes enzyme inhibition during downstream reactions (e.g. ligation, PCR). It may be that the agarose is old and the quality is no longer good or it may brand-dependent. We can’t provide a concrete reason for this observation, but bear in mind that simply switching to a different bottle of agarose may increase your chances of cloning success.
5. Run Controls
To determine if your problem is actually related to the gel extraction process, try running a control in which you digest empty vector with a single enzyme, perform the gel extraction, and re-ligate it. A vector cut with one enzyme should re-ligate very easily and provide plenty of bacterial colonies on a transformation plate. If the control ligation works, then the inability to clone your DNA construct may be related to some other factor, such as secondary structure of the DNA, repeat sequences causing instability in E.coli, or the DNA may encode a protein that is toxic in bacteria.
The Following Tips Apply If You Are Using Commercial Silica Spin Kits
6. Renature the DNA
The melting step combines high concentrations of chaotropic salts with heat. This combination will denature the DNA. If the eluted DNA appears at half the expected size (it is now single-stranded), renature the DNA by warming it up to 95°C for 1 minute and let it slowly cool down to room temperature.
7. Wash It Again
An extra washing step with the ethanol-containing wash buffer in the kit will always help get rid of chaotropic salt residues on the membrane. Washing is a critical step in the gel extraction process since carryover of chaotropic salts will inhibit DNA ligase.
8. Make Sure All of the Ethanol Is Gone
The silica membrane must be dry after the ethanol wash steps to ensure a good yield and successful cloning. To determine if you have ethanol in the final DNA, run a test agarose gel on the eluted sample. If the sample floats out of the well (even in the presence of loading dye), you have ethanol contamination. To enhance the drying step (especially if you live in areas where humidity is high), try centrifuging the spin column with the cap open to maximize airflow through the membrane.
9. Only Use High-Quality Ethanol
It is critical to use high-quality ethanol in the wash buffer and not denatured alcohol. Denatured alcohol contains chemicals such as isopropanol, methanol and benzene, and these chemicals will not dry from the silica membrane, thus making their way into your DNA solution. You know you’ve used denatured alcohol if you ever notice that a) your DNA smells funny, b) your DNA won’t freeze at –20°C, c) you observe the floating phenomenon mentioned above.
10. Elute Your DNA With Hot Elution Buffer
Heating the elution buffer to 70°C before applying your sample to the column will release more of the DNA from the membrane, resulting in higher yields. Allowing the buffer to sit on the column for 5 minutes before centrifugation can also help to increase yield.
11. 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!
12. 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.
13. 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.
14. 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.
15. Guanosine – Sunblock for DNA.
We know how damaging UV light can be to 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.
DNA Gel Extraction Tips Summarized
Extraction of DNA from a gel is an essential step in most cloning and sequencing projects. It doesn’t have to be the bottleneck for getting to the real work of expressing the protein or genotyping your DNA. With these simple tips, gel extraction will be a walk in the park, sending you on your way to ligation success in no time!
Want an alternative to kits for your DNA gel extraction? Discover old-school DNA gel extraction methods that can save you money and reduce waste.
Originally published on November 12, 2007. Updated and revised in May 2017.
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