EZ-Gels

A project Log for building simple electrophoresis apparatus for DNA analysis.

Gel electrophoresis has an indispensable tool for analysis and preparation of DNA since the early days of recombinant DNA technology in the 1970's. While there have been incremental improvements over the years the tools and methods, not all have been well explored for low-cost DIY biology experiments. This section describes attempts to explore practical approaches to make electrophoresis cheaper, better and more accessible. Like much of the content of this website, this is work in progress and I focus on the current best performance of the devices and building instructions. There will always be room for improvements and hopefully some of this will be useful as a springboard at least.

Agarose gel electrophoresis has been the mainstay for analysis of ~100bp-20kb DNA fragments. Research grade electrophoresis equipment generally costs in the range of £200-£800. There have been some notable attempts to provide low cost systems for education (e.g. Edvotek, Minipcr, Biorad, NCBE), but arguably there is still room for cost reduction, improved performance and flexibility through incorporation of technical advances that have emerged in the research community. These include the use of:

1. 3D printing to create custom electrophoresis vessels, dams and combs.

2. Electrically conductive filament to print plastic electrodes, as an alternative to expensive platinum wire.

3. Incorporation of standard glass plates as a flat, transparent base for pouring the gel.

4. Low ionic strength buffers to reduce resistive heating and speed migration rates.

5. Minimisation of the size of the device without compromising resolution for easy handling.

6. Integrate blue LED illumination and long-pass emission filters for direct observation of in-gel stained DNAs and avoidance of UV-mediated DNA damage that comes with shorter wavelength excited dyes like ethidium bromide.

7. Future use of ion exchange resins as a source of ions for buffer-free electrophoresis (similar to commercial E-Gel cassettes).

This page contains a summary of trials to incorporate some of these features into a practical device. Some of the prototype devices are shown (below left) - along with a diagrammatic version of the current best performing favoured design (below right).

Design features

1. 3D printed tank. I chose to use 3D printing to build the main body and accessories for each gel apparatus. PLA filament was used for reliable printing and low cost. The choice of 3D printing gives the benefit of highly flexible design and assembly, but comes with a few issues that need to be tackled. Can the gel tanks be made reliably water tight? How to integrate the electrodes and connection to a power supply? All of the parts have been printed with generic PLA filament (1.75mm on a Bambu Lab printer with 0.4mm nozzle and 0.2mm layer depth) with 100% infill. The printed tanks have 4mm thick walls and show no signs of leakage. Further the use of 3D printing allows adjustment of the design to ensure that the dams and combs have matching slots in the vessel, and can be adjusted for close tolerance fitting. This makes the apparatus very easy and reliable to use - no leaks! The standard PLA material has a useful surface - slightly hydrophobic so that the the dams and combs can be easily removed after gel set without problems of gel adherence. The comb teeth are printed with 1.5% sloping lateral edges to ease removal without damaging the wells in the gel.

2. Embedded glass base. Ideally, the gel needs to be poured onto a flat, transparent surface with removable comb and dams. I decided to base the design on glass microscope slides that can be embedded in the tank design. These are made in 75x25mm and 75x50mm sizes. I tested different shaped gel tanks with glass slides in different sizes and orientations (some examples as shown above left). The best solution was to use the 75x50mm slides, with the direction of electrophoresis across the long dimension. The larger size slides are harder to find than the ubiquitous standard size, but can be obtained from microscopy specialists or on Ebay or Aliexpress. The glass slides are available with 1-1.2 mm thickness - this should be checked with a micrometer for batches from different suppliers. The gel boxes are printed with a precise inset to accept the glass slide. This has a 0.2mm tolerance on each side and has a 2mm shelf to support the glass. The glass slides are permanently fixed into the gel box with glue, using a fine application around the shelf. This has proved simple and waterproof.

3. Anode and cathode reservoirs. I was initially looking to build minimally-sized gel cassettes that employed ion exchange resins as buffer-less reservoirs, similar to commercial E-gels. In this, I was guided by published patent literature (WO 97/41070, Apparatus and method for electrophoresis Cabilly et al, 1997), which describes the clever use of complementing weak ion exchange resins for minimal anode and cathode reservoirs. However, my trials ran into problems with ion and pH balance and electroendo-osmosis. This is probably a challenge worth returning to, since the commercial option works! In the meantime, I reverted to a more conventional solution with small-size liquid buffer tanks.

4. Low ionic strength electrophoretic media. TBE and TAE buffers have been the default buffers used for agarose gel electrophoresis, with discussion about their relative benefits for DNA separation and effects on subsequent DNA manipulations for isolated fragments (e.g. Goldbio and BitesizeBio). As Brody and Kern have pointed out the original use of these buffers was more driven my existing precedent than best choice. A good overview of this can be found in Brody, JR and Kern SE (2004) History and principles of conductive media for standard DNA electrophoresis. Anal Biochem. 333(1):1-13. They have demonstrated that lower ionic strength buffers like several millimolar sodium or lithium borate solutions provide faster electrophoretic separations with lower resistive heating at higher voltages (Brody, JR and Kern SE (2004) Ultra-fast high-resolution agarose electrophoresis of DNA and RNA using low-molarity conductive media. Biotechniques 37(4):598, 600, 602 and Brody, JR and Kern SE (2004) Sodium boric acid: a Tris-free, cooler conductive medium for DNA electrophoresis. Biotechniques 36(2):214-6). One potential pitfall with these low strength media is that they have very little buffering capacity, so that relatively large cathode and anode tanks. or media recirculation is required to avoid ion depletion or pH shifts due to electrolysis. This is a problem for the mini-gel cassettes described here. Happily, there is a compromise solution. Sanderson et al. showed that the omission of disodium EDTA from TBE and use at half-strength provides some of the benefits of low ionic strength, while still retain Tris and borate as buffering ions (Sanderson et al. (2014) Modification of gel architecture and TBE/TAE buffer composition to minimize heating during agarose gel electrophoresis Anal Biochem. 454:44-52). This 0.5x Tris-borate (TB) buffer works well with the mini-gel cassettes.

5. Plastic electrodes. The use of 3D printing to build gel tanks has been given a boost by the recent emergence of electrically conductive filaments. I used Protopasta Electrically Conductive Composite PLA filament (https://proto-pasta.com/products/conductive-pla), which is an easy to print PLA with embedded carbon black. The printed material has a resistance of 4.8-8.4 Ohms/cm along a filament, and printed parts can have 30 Ohms/cm within a layer and 115 Ohms across layers. So, the printed material has significant resistance compared to a metal conductor. However, the solution-facing part of a 3D printed plastic electrode can provide a broad face for formation of an even electrical field. I have also used conductive copper adhesive tape as a backing, to minimise the resistance between the electrode and contact with the power supply, and ensure an even and minimum path for current flow.