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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.
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)
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Is making up a solution of 5mM lithium acetate all I have to do to run my gel? How about the pH?
Some say to add EDTA to a final concentration of 1mM, does anyone know whether it make any difference?
I’ve been using boric acid gels for several years, and love them, but just found out boric acid is now a suspected teratogen: may harm the unborn child. Also other potential toxicity concerns. I’m probably going back to TAE.
On the boric acid package there are similar warning.
Most applications in our group require the addition of MgCl2 in order to stabilize the DNA in specially folded structures.
Does anyone of you think there might be disturbing effect by mixing Sodium Borate with Magnesiumchloride?
Thanks in advance for your opinion!
It is my time to try SB. My gel is 13 cm long. I tried 1.5% gel and 3% gel at 150V for 2 hours and 2% gel at 200V for 3 hours. My samples’ performance were even worse than on TBE and TAE gel. But it might be caused by samples. Let’s just say the ladders. The ladders I used were Bioline Hyperladder I and IV.
1.5% gel: 100bp was at ~8cm; 1000 bp was at ~3 cm; >1500 bp were stuck together
3% gel: 100 bp was at ~4cm; >500bp were stuck together
2% gel: 100bp was ~11 cm; 1000 bp was at ~4 cm; >1500 bp were stuck together
at 150V, I did not feel hot.
at 200V, the gel was hot.
SB is good for small size DNA, epically for DNA <500 bp, for two reasons: 1) time saving, 2) good resolution.
for big size DNA: 1) time saving is not significant, 2) resolution is bad.
I’ve similar experiences. Nevertheless it’s works fine for PCR product control. 3% agarose gel electrophoresis with SB now takes about 27 min. to compltete at 300V :). The only problem is that orange G migrates very quick now – far faster than in TAE/TBE buffer – so when it comes to the end of ca. 10 cm gel 50bp band is barely 5 cm from the start. Bromophenol blue is a bit better.
Any of you have any recommendations for replacement?