Quantcast

Established in the mid 1970's, New England Biolabs, Inc. (NEB) is the industry leader in the discovery and production of enzymes for molecular biology applications and now offers the largest selection of recombinant and native enzymes for genomic research. NEB continues to expand its product offerings into areas related to PCR, gene expression, sample preparation for next generation sequencing, synthetic biology, glycobiology, epigenetics and RNA analysis. Additionally, NEB is focused on strengthening alliances that enable new technologies to reach key market sectors, including molecular diagnostics development. New England Biolabs is a privately held company, headquartered in Ipswich, MA, and has extensive worldwide distribution through a network of exclusive distributors, agents and seven subsidiaries located in Canada, China, France, Germany, Japan, Singapore and the UK. For more information about New England Biolabs visit neb.

Faster, Even Cooler DNA Gels!

Content brought to you by New England Biolabs

When I began a master’s program in 2008, the lab task I hated more than anything was running agarose gels for DNA. Something so simple, ubiquitous, and necessary gobbled up more hours in the lab than I care to remember. Even though we added the DNA stain directly to the molten agarose and didn’t have to spend time staining gels, gel electrophoresis still ate up entire days in the lab.

I figured that there had to be a faster way to do this.

A way to resolve small bands quickly

While waiting for one of my many gel runs to finish, I spent some quality time with Google to find a solution to my problem. I was introduced to Bitesize Bio for the first time when I came across this article, “Faster, Cooler DNA gels”. By using sodium borate (SB) electrophoresis buffer, you can crank up the voltage on the gel box without worrying about distorted DNA bands or even that the gel will melt. The payoffs are good resolution of small DNA fragments (~ 100 bp – 3 kb) and time saved with a shorter gel run. For example, a gel run in SB takes 25 minutes minutes vs. 45 minutes with TBE.

If you want to really get fancy, you can use 1 mM lithium borate (LB) to resolve fragments even smaller than 100 bp.

Being able to run my gels faster with a borate-based electrophoresis buffer was a revelation and allowed me to get much more done in the same amount of time. Why didn’t everyone know about this??

A snag – it doesn’t work with larger DNA fragments

Most of the time, when I wanted to run a gel to look for a band in the neighborhood of 300 bp – 2 kb, SB worked great. However, if I needed to gel-purify a vector, things got dicey. All of the bands of interest – cut vector, uncut vector, ladder, anything bigger than 4.5 kb – were fuzzy, diffuse, and just about useless. Even when I turned the voltage down to the speed of a moderately ambitious snail.

One big drawback that SB and LB buffers share with their cousin, TBE, is poor resolution of large DNA fragments. I learned this the hard way. Several times, in fact.

Lithium acetate (LA) buffer is the voltage-cranking equivalent to TAE, allowing quick resolution of large DNA fragments. This became my solution for gel-purification of vectors, but the downside here is poor resolution of small DNA fragments.

You can have it both ways!

So I went back to Google. I may even have used Google Scholar this time – that’s how serious I was about finding a single buffer that could handle the range of DNA sizes I worked with. In my case, I needed more resolution on the high end than the low end. Eventually I found this paper, which describes what amounts to a combination of LB and LA buffers.

Duh! Why hadn’t I thought of that?!

It took me several reads before I teased out the composition of lithium acetate-borate (LAB) buffer. In addition, this website lays out the recipe very nicely.

25X LAB buffer

  • lithium acetate   16.5 g   (250 mM)
  • boric acid           15.46 g (250 mM)
  • ddH2O                 to 1 L
  • Make sure the pH is between 6.5 – 7.0

How to run a LAB gel

As with other agarose gels, you should make the gel with the same running buffer you plan to use during electrophoresis, which is 1X LAB. I typically run my gels at about 30 V/cm for 20 – 30 minutes. As you can see in this picture of a 1% gel made in LAB + GelRed, even the high molecular weight bands are pretty well resolved. Unfortunately, my DNA didn’t show up, but the DNA markers are gorgeous!

decent resolution

Separation of bands in 1% agarose gel made using LAB buffer. I used the 1 Kb Plus DNA Ladder (Invitrogen) here.

 

Last minute tips:

  • You can cast agarose gels in advance to save on prep time. Just store at 4°C in some running buffer until you need them.
  • For sharper bands, try using Ficoll instead of glycerol in your loading buffer. Your sample stays in the bottom of the well better and doesn’t start to diffuse before you have finished loading your gel.

If you like the idea of fast gel runs and awesome looking DNA bands regardless of molecular weight, you might want to give LAB buffer a try.

References

1.  Singhal, H. et al. (2010) Improved DNA electrophoresis in conditions favoring polyborates and Lewis acid complexation. PLoS ONE 5:e11318.

2.  Brody, J. R. et al. (2004) Ultra-fast high-resolution agarose electrophoresis of DNA and RNA using low-molarity conductive media. Biotechniques 37:598-602.

12 Comments

  1. vishal on March 26, 2019 at 7:49 pm

    I tried the recipe at 2.5 X voltage and compared it against TAE run. The run, of course, got complete more or less 2.5X quicker (expected) and the gel though was hot, would certainly melt if the buffer was TAE. However, when it comes to separation, the results were not that impressive. I used a ladder that spanned 75bp-20kb (Generuler 1kb plus) and TAE was clearly better at resolving both ends of the sizes, especially at the higher end. In LAB buffer the bands were thicker AND less separated making it difficult to estimate size. I also saw that adding salt (that would be in line with restriction or PCR buffers) shifted bands by ~15% of size.

  2. Daniel on March 17, 2019 at 2:30 am

    I’ve tried this twice now, and my gels melt. There’s still too much heat being generated at these high voltages. 🙁

  3. amin on November 15, 2018 at 9:30 pm

    hi, since this buffer doesn’t contain EDTA can nuclease contamination become a problem?
    kind regards

  4. AND on October 13, 2018 at 11:31 am

    Can i substitute boric acid with borax?

  5. Christian on July 10, 2018 at 5:42 pm

    Hi I have a question,
    does the unit “V/cm” refers to the length between electrodes or the length of the gel?

  6. Lodoiska on June 21, 2018 at 9:51 pm

    Hi!
    I love this recipe to run gels. I compared with TAE and TBE, and the heating difference is tremendous. So thanks!

    I have to ask though… do you really run your gels at 30v/cm?? Or is it a typo?

    Thanks!
    Lodo

    • Ian Hu on August 16, 2018 at 12:32 pm

      The 2004 Kern paper ran some of their LAB at 1000V (100V/cm). A. Thousand. Volts. No 30v/cm is not a typo.

  7. Linda on January 16, 2018 at 5:22 pm

    Cool! Thanks for putting this together.

  8. Howdy on October 16, 2015 at 5:07 pm

    The website you link to presents a different recipe for the same 25X buffer: –

    For a 1L 25x Solution

    25.5g Lithium acetate dihydrate
    15.5g Boric acid
    make up 1L with dH2O

    Which one is it?

    • Kristie Mitchell on October 18, 2015 at 1:58 am

      The answer is both! My lab uses anhydrous lithium acetate, where 16.5 g will give a final concentration of 250 mM in the 25x buffer. The recipe you listed uses lithium acetate dihydrate, where 25.5 g will also give a final concentration of 250 mM. To the best of my knowledge, there is no difference in the performance of the buffer whether the dihydrate or anhydrous lithium acetate is used.

  9. Caroline on September 23, 2015 at 4:01 am

    when I tried the original faster better media system out there were some problems running restriction enzyme digests direction on the gel, something about the high salt content in the reaction. Is this a problem with this hybrid system?

    • Kristie Mitchell on September 23, 2015 at 4:31 pm

      I have not noticed any problems running restriction enzyme digests directly on a gel. That is typically how I purify cut vectors for ligation and I get well-defined bands.

Leave a Comment





This site uses Akismet to reduce spam. Learn how your comment data is processed.

Share via
Copy link
Powered by Social Snap