Cargo Cults and Electrophoresis

Toward the latter half of WWII, landing strips and airbases were being built all over the Pacific on small, otherwise totally isolated islands. Little contact was made with the natives to explain what was going on, and in some cases the locals were incredulous to see the new arrivals performing strange rituals on their tarmac runways and receiving airdropped “gifts” from the gods/ancestors.

When the bases were abandoned, in some cases the locals started replicating the structures and activities of the base personnel, performing marches and drills, talking into replica radios, and waving lit firebrands on the runways and waving them. Elements of Christianity were often woven into the practise in an attempt to attract the same blessings as the military staff had. These culturally contaminated pseudoreligions are called “cargo cults“, and these words have since been applied to any misapplication of a procedure to an end without understanding how it works.

It might be premature of me to describe modern Electrophoresis as it is performed in most labs as “Cargo-cultish” until I’ve properly demonstrated that I have a better alternative, but having read this paper over coffee this morning, I was really reminded how little me or my fellow lab staff really bother to understand Electrophoresis, a fundamental, essential part of our daily work with DNA and the part whose quality is usually what ends up being scrutinised on peer reviewed papers!

Ask someone which buffer to use and you’re always offered one of three options: 1x TAE (Tris-Acetate-EDTA), 1x TBE (Tris-Borate-EDTA) or 0.5 TBE for those edgy folks who’ve been doing this for a while. TBE offers better resolution when you go to look at your results, and can be run at higher temperatures, so people often favour TBE for routine use. Some people in the know will suggest TAE where you’ll need to do something to the DNA afterwards, because the Borate in TBE is high enough concentration that it can mess up DNA extractions from the Gel and enzymatic reactions later. I only learned this upon reading the aforementioned paper, and I’ve frequently lost my DNA upon gel extraction, which I now realise may be down to my selection of buffer.

As the paper intricately explains, the use of Tris buffers is a holdover from DNA electrophoresis being borrowed from protein electrophoresis. What followed after its adoption was a brief spree of experimentation, followed by a trend towards standardisation that lead to everyone adopting TAE or TBE so they could all understand one another’s protocols. The assumption was that any effort put into improving gel resolution was wasted effort, as only marginal gains would be made.

However, there is a big problem with Tris buffers that haunts anyone performing Electrophoresis and causes all manner of problems, even when it isn’t recognised to be the cause of these problems. Heat generation due to the current passing through the gel can cause the gel to soften or even melt, and worse still as the heat increases the conductivity of the gel increases also, leading to runaway heating and poor outcomes.

Problems caused directly or indirectly by heat include:

  • “Smiling” bands,
  • Smeared or blurred bands due to uneven softening of gel surface
  • Band diffusion, forcing one to perform oversized gel extractions
  • Total Gel Meltdowns
  • Having to run at low voltages and missing dinner.

The reason behind this runaway reaction is the inclusion of Tris and excess Sodium from the preparation of the EDTA used in the buffer.

EDTA, they argue, isn’t even needed nowadays. Apparently they ran a gel using creek water without EDTA and had few issues; the enzymes used today have few unwanted activities under electrophoresing conditions. Or so we are told.

Tris, meanwhile, is too conductive and permits a lot of current through the Gel at a given voltage. As the gel heats, it lets even more through.

The authors experimented instead with a simple electrophoresis buffer composed of Sodium Borate at 10mMol (among other experimental buffers) and found it to be ideal on almost all fronts; Sodium Borate is cheap, can be prepared as a buffer from only one crystalline ingredient, gives excellent resolution, heats very slowly and can be used at very high voltages without issue (meaning faster gels).

If this is true, it means that Gel Electrophoresis as it is commonly performed is not only flawed but overpriced; a quick check of the price of 1L 10x TBE buffer from Bioplus shows that it costs $12.75. For 17.49/kg from the Science Company, I can make 52L of 10x Sodium Borate (1x SB Buffer is 1.907g/L Disodium Borate Decahydrate, the usual crystal form).

So the costs per litre of the two are $1.28 for TBE, and $0.04 for SB. For your investment of 4c you get better gel resolution at higher voltages (meaning less time waiting), which can apparently be more reliably purified and used downstream for enzymatic reactions such as ligation or PCR.

This is great if it’s true in my labwork at the CCRC, but it’s even better news for DIYbio folks worldwide: You can often find Sodium Borate on ebay where you’ll never see TAE/TBE, and the cost difference is pretty staggering.

More on this when I next run a gel: I have a bottle of 5x and 1x SB buffer next to me, just waiting for me to add Agarose and give it a try.

3 thoughts on “Cargo Cults and Electrophoresis

  1. Interesting you made your own! The 20x sample we include with the Pearl Gel Box is courtesy of these guys (the authors of the paper you cited too!): http://fasterbettermedia.com/

    In terms of pricing, they sell 1 liter of 20x SB for $42.50.

    Looking forward to seeing your results with SB.

    Tito

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