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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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Can anyone comment on their experiences using lithium boric acid buffer to resolve short (<40 nts) DNA and RNA? I routinely use TBE in a 20% PAGE gel and good results, but in hours. I’m hoping to speed up the analysis by using the new buffer system.
I guess this a bit of a follow up to Brian Cady’s question – I’ve used some old Na2B4O7 x10H2O that we had around the lab and that gives pH around 9.4… I’m assuming 20g borax/1L H2O gives pH that’s still a bit off. Do you just adjust pH with boric acid? the elevated anion concentration does not affect the gel properties?
My lab only runs the sodium borate gels now. We used to run them at 350V but burned up a power supply so now we only run them at 250V.
I use the gels from 0.7% to 4.0% depending on the purpose. The 4% gels end up running about 20 minutes for good resolution and the buffer gets hot but I have never melted a gel.
The best ladder we have found is the Fast DNA Ladder from NEB. All the others we tried would smear very badly but this one runs beautifully.
I’ve also used the Lithium acetate buffer to resolve topoisomerse of intact plasmids. You need to add 1 mM EDTA and pH it with LiOH. Unfortunatly high voltages seem to make the plasmids run strangely (maybe there adopting a new conformation?). The resolution at lower voltages is great though.
When adjusting the 20X SB stock solution pH, does one use solid boric acid? I tried preparing 0.5M boric acid solution, but its pH is only around 4, and the pH of the SB buffer has barely budged with 15 ml of 0.5M boric acid solution added to 500ml of 0.1M disodium tetraborate solution. This has a pH of about 9.4 originally, similar to what was noted above.