What is it about gel extraction of DNA that makes it a pain? Maybe it’s poor product yields or maybe it’s because the process uses harsh chemicals (chaotropic salts, ethidium bromide, ethanol, heat) that will damage or denature DNA and potentially decrease cloning success. In this article I share some tips, both from experience and from helping people with the procedure, to help maximize yields of high quality DNA from the gel extraction process. I hope these suggestions help you to obtain high yields and purity of double-stranded DNA.
1. Trim the gel slice as much as possible. Get rid of all the excess gel including in front of or behind the DNA. Most people cut out a square around the gel but don’t think to stand it up and trim the gel on front and back. If you poured a thick gel, there will be a lot more gel to remove. The more you can remove encasing your DNA, the higher the yields.
2. Minimize exposure on the 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 on the UV. Don’t let the entire gel sit cooking on the UV light while you cut one slice at a time. The DNA sitting the longest will be nicked to shreds. Alternatively, use a visible range stain such as methylene blue or crystal violet. More info in this article on vector preparation tips.
3. Remove all traces of phenol using a “home brew” method. If using phenol to purify the DNA from agarose, carry-over of phenol will not be removed by ethanol precipitation and will inhibit the ligation. To remove traces of phenol from the aqueous phase, warm the supernatant at 65C for 5 minutes to evaporate. Let it cool back to room temperature over 10-20 minutes before precipitating to make sure you have double stranded DNA.
4. Change to a new brand or bottle of agarose. Sometimes, for some reason, agarose actually causes enzyme inhibition. It may be that the agarose is old and the quality is no longer good or may be certain brands. I can’t say for sure, but I have seen cases where simply switching to a different bottle of agarose results in cloning success.
5. Run controls to determine if the problem is actually the gel extraction step, try running a control where you digest empty vector cut 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 colonies on the plate. If it does, then the inability to clone the DNA may be related to some other factor, such as secondary structure of the DNA, repeat sequences causing instability in E.coli, or the DNA cloned codes for a protein that may be toxic in bacteria.
The following apply if you are using commercial silica spin kits:
6. Renature the DNA. The melting step combines high amounts of chaotropic salts with heat. This combination will denature the DNA. If the eluted DNA appears half the expected size (it is now single-stranded), re-nature the DNA by warming up to 95C for a minute and let cool slowly to room temperature.
7. Wash it again. An extra wash step with the ethanol containing wash buffer in the kit will always help get rid of chaotropic salt residue on the membrane. Carry over of the salts will inhibit ligase.
8. Make sure all of the ethanol is gone. The silica membrane must be dry after the ethanol wash step to ensure a good yield and for cloning. To determine if you have ethanol in the final DNA, run a check gel on the eluted sample. If it floats out of the well (even with 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 air flow through the membrane.
9. Make sure your ethanol is the good stuff. It is critically important to use high quality ethanol in the wash buffer and not denatured alcohol. Denatured alcohol contains chemicals like isopropanol, methanol, and even benzene and these chemicals will not dry from the silica membrane and will carry into the DNA. You used denatured alcohol if you ever noticed that a) your DNA smells funny, b) you DNA won’t freeze at -20C, c) you observed the floating phenomenon mentioned above.
10. Elute with hot elution buffer. Heating the elution buffer to 70C before applying it 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.
Extraction of DNA from a gel is a necessary part of 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 DNA. With these simple tips, you will be on your to ligation success!
Photo: Axel Houston