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Faster, Cooler DNA gels
Content sponsored by New England Biolabs
All over the world, molecular biologists are tragically wasting hours of their life running DNA gels using tris-based conduction buffers like TBE or TAE.
These buffers are known to overheat at high voltages, causing problems with gel integrity, sample denaturation and more. Because of this, molecular biologists are forced to keep the voltage of their gels to a maximum of 5-10 volts/cm (e.g 100 volts for a 10 cm gel) and extend the running time, sometimes to hours.
Although long gel runs, like long restriction digests, are often used as a convenient coffee break opportunity they can also eat into the molecular biologist’s precious time, leading to longer and less efficient working days.
But, in 2004, a team of scientists from Johns Hopkins came up with solutions (pardon the pun) to this problem. They have developed and verified three conductive buffers that stay cool during electrophoresis, allowing the voltage to be racked up to a massive 35 volts/cm without any problem, reducing the time taken to run gels by up to 7 times.
Between them, the three buffers cover all of the molecular biologist’s DNA gel needs. The buffers are:
- 10mM sodium boric acid (Na2B4O7/Borax)
For standard applications (separation of DNA fragments from 100bp-5kbp). - 5mM lithium acetate (LiOOCCH3, CAS:546-89-4)
For separation of fragments longer than 3 kbp. - 1mM lithium boric acid (Li2B4O7, CAS:12007-60-2)
For separating small DNA fragments and ssDNA
Our lab’s standard DNA gel buffer has become 20 g of Borax into 1 l of water which gives a 20x stock solution. Small gels run in 10-15 minutes at 200V.
The sodium borate and lithium acetate buffers can also be used for RNA gels in place of MOPS buffer.
Simply make up the required buffer, use the same buffer in the gel and the tank, turn up the voltage to 10-35 volts/cm and watch that DNA go.
Despite this excellent work, there are still thousands of molecular biologists who have not yet been shown that there is an alternative to wasting their precious hours using tris buffered gels.
Help us to eliminate this tragedy by spreading the word about this method for faster, cooler DNA gels. You can do this by clicking the link below this article to e-mail it to your molecular biologist friends. We, and they, thank you for your help.
Further reading:
1. Brody and Kern(2004) Biotechniques 36 p214
2. Brody et al (2004) Biotechniques 37 p598 (Free registration required)
3. Hudson, Biocompare protocols (a protocol giving a quick overview)
Photo: Klis
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I really like this buffer system very much, except for the following issues:
It is REALLY sensitive to salts and glycerol, so if you’re doing the classic alkaline lysis miniprep and restriction digests, you will get substantial gel retardation problems unless you either dilute your sample or clean up a column, which in my opinion defeats the purpose of cost and time savings, not to mention that the ladders become somewhat useless. That being said, it is GREAT when analyzing PCR fragments as the resolution is very good and migrates well with the ladders. I use 5mM final concentration of Borax (20X stock is 100mM which is close to the limit of solubility). Lastly, because of the time savings, I’ve started to tinker with the boiling miniprep protocols as they have substantially less salt in them and it looks fairly promising to combine with the Borax buffers.
I have not tried yet the LiOAc but will be doing that soon as most of what I have been analyzing is lower than the 3kb limit.
Ivan Ivanov added the following by mail, when I asked him:
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I will try to explain in more details:
The optimal final concentration of Na+ in the buffer should be around 10mM and lower, according to Brody et al. 2004.
There are several different forms of borax on the market. The one that you use, seems to be the anhydrous (waterless) form with molecular weight 201.2 (and molar mass 201g/mol). That’s why if you dissolve 20.1g in 1L you will obtain 100mM solution of borax but 200mM of Na+, since the formula is Na2B2O7. From here I am sure you can do the math.
There is one more thing… my experience is that borax is very difficult to dissolve (use boiling water) and gives pH around 9.5. That’s why I always add boric acid to pH 8.3.
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Ivan,
Glad you have had success with this! Thanks for pointing out the typo – I have fixed it now 🙂
Isn’t 40 g/L correct? I believe borax is is the decahydrate version (Na2B4O7*10H2O) which would correctly make it approximately 10 mM Na+.
Hi, guys
I just wanted to express my excellent experience with these media. I have obtained very good resolution on 3% standard agarose gels (2 bp for microsatellites).
There is a mistake on the above recipe for the SB !!! If prepared according to the above (40g/l borax for 20x) you will end up with 20mM Na+ final conc. (instead of 10mM) as 1 mol of borax (Na2B4O7) provide 2 mol of Na+.
Isn’t 40 g/L correct? I believe borax is is the decahydrate version (Na2B4O7*10H2O) which would correctly make it approximately 10 mM Na+.
Thanks for the link Caroline! The FAQ says that if you use HIGH salt restriction digest buffers, you’d better dilute them a little before putting them onto a gel. As SBA/LB are lower salt than TBE/TAE, the salt difference samples/gel starts to play a role.
It seems that I never used high salt buffers until now with the gels so I cannot say how strong the effect is.
I guess it is a good idea in general, also for TBE, to dilute the ladder with the buffer of the samples you’re running and, in the case of LB/SBA, to dilute the samples with the running buffer a bit.
Has anyone experienced this? Has anyone calculated which of the standard buffers (e.g. NEB 1-4) has a salt content > 50 mM ?