What is it about DNA gel extraction 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 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 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. You can read more about this in our article on preparing vectors for cloning.
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 65°C 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 it may be certain brands. I can’t say for sure, but I have seen cases in which 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 in which 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 95°C 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 successful cloning. To determine if you have ethanol in the final DNA, run a check gel on the eluted sample. If the sample 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 know you’ve used denatured alcohol if you ever notice that a) your DNA smells funny, b) you DNA won’t freeze at –20°C, c) you observe the floating phenomenon mentioned above.
10. Elute with Hot Elution Buffer
Heating the elution buffer to 70°C 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 way to ligation success!
Originally published on November 12, 2007. Updated and revised on March 18, 2016.
Over the past few decades molecular biologists have developed procedures to simplify and standardize cloning processes, allowing vast arrays of artificial DNA structures to be more easily assembled. Are you familiar with all the cloning options out there? Let’s look at five different cloning methods you can use to get your construct. At the end […]
It’s great to have you in the Bitesize Bio family! We’ve sent you an email to confirm your registration. Please click on the link in the email or paste it into your browser to finalize your registration.
For more information on how to use Bitesize Bio, take a look at the following image (click it, for a larger version)
An error occured while registering you, please reload the page and try again