This section provides an overview of horizontal electrophoresis, gel boxes, running buffers, and agarose types, as well as discusses some factors affecting resolution of DNA fragments and suggests appropriate agarose types and concentration for different sizes of DNA molecules. Pulsed field gel electrophoresis is discussed in a separate section.
Gel boxes are available in different sizes to accommodate different needs. Mini formats are usually used for quick separations and a small number of samples; large formats are ideal for restriction fragment length polymorphism (RFLP), Southern blotting, and PCR sample screening. Bio-Rad offers multiple choices of gel boxes called Sub-cell® systems to fit different applications.
Many users cast their own gels; in fact, gels can be cast either directly in the gel box, using a gel caster, or in a UV tray on which the openings are closed off by adhesive tape. A large selection of tools, including combs with different numbers of wells and different well thicknesses, or different sized UV trays and gel casters, are available for simplifying gel casting.
For convenience, many users purchase precast gels, which are available in different sizes and gel percentages and are prepared either with Tris-acetate-EDTA (TAE) or Tris-borate EDTA (TBE) buffer. Bio-Rad offers numerous precast gels and ReadyAgarose™ gels for your convenience.
Bio-Rad's Sub-Cell® System Selection Guide
* Mini ReadySub-Cell GT is a Mini-Sub cell GT dedicated to running ReadyAgarose precast gels, gel size 7 x 10 cm; sample throughput is 8, 12, or 2 x 8. This cell does not include casting gates, tray, or combs. ** Wide mini ReadySub-Cell GT is a wide Mini-Sub cell GT dedicated to running ReadyAgarose precast gels, gel size 15 x 10 cm; sample throughput is 20, 32, 2 x 32, or 4 x 26. This cell does not include casting gates, tray, or combs. *** Sample throughput value assumes 1–2 combs per gel. † Sample throughput value assumes 1–4 combs per gel.
Optimal Resolution Range of ReadyAgarose™ Gels
* A 3% ReadyAgarose gel is easier to handle and gives the same performance as a standard 4% gel.
Handcast Agarose Gels Handcasting offers options for a variety of gel sizes. Two accessories are available for handcasting gels within the Sub-Cell family of electrophoresis cells. Gel casters offer the flexibility of accommodating several different-sized UV transparent (UVTP) gel trays and are available for the entire Sub-Cell product line. Casting gates offer the convenience of casting directly in the electrophoresis cells and are available in one specific size for each cell (see Casting Guide below).
Hand Casting Options for a Variety of Gel Sizes. Two accessories are available for hand casting with the Sub-Cell family of electrophoresis cells: gel casters and casting gates. Gel casters offer the flexibility of accommodating several different-sized gel trays. These are available for the entire line of Sub-Cell systems. Casting gates, available in specific sizes for each cell, offer the convenience of casting directly in the Sub-Cell cells.
An electrophoresis buffer conducts an electric current across the electrophoresis chamber; the most commonly used buffers for DNA electrophoresis are TAE or TBE. For full recipes see table below.
Linear double stranded DNA is separated faster in TAE buffer; however, this buffer has a lower buffering capacity than TBE because the borate in the TBE buffer acts as an enzyme inhibitor and could negatively impact downstream applications. Therefore, TAE is the preferred buffer if the DNA will be used for cloning or ligation.
Electrophoresis Buffer Selection Guide
Blotting Buffer Selection Guide
* These buffers can be used for all gel types and formulations.
A variety of agarose powders are available. When selecting the type of agarose to use, the most important factor to consider is the size of the fragments being separated. In addition, considering the percentage of agarose in the gel may better resolve the fragments of interest and produce a gel that will yield a publication-quality image. Higher percentage gels resolve smaller DNA fragments, whereas lower percentage gels resolve larger fragments. Each type of agarose in Bio-Rad's Certified™ Agarose product line is genetic quality tested (GQT) and can be used for routine separations and downstream molecular biology applications.
Nucleic Acids Stains and Tracking Dyes Nucleic acid molecules are colorless and a tracking dye such as bromophenol blue or xylene cyanol is generally used to track their movement in the gel. After electrophoresis, nucleic acids are stained. The fluorescent stain ethidium bromide is the most commonly used, but other stains are available. After staining, the gel is visualized using a gel imager.
Routine SeparationsCertified molecular biology agarose is recommended as a general-purpose agarose for routine separations of ~500 bp to 20 kb DNA fragments. This agarose offers rapid migration rates, easy-to-manipulate gels, and displays high strength even at low agarose percentages.
Influence of gel percentage on optimal DNA fragment resolution
Separations of Small FragmentsFor high resolution separations of small fragments such as PCR amplified fragments, Certified PCR agarose is recommended. This agarose separates DNA fragments from 20 bp to 1000 bp. The standard gelling temperature makes it easy to prepare, and the gels are easy to manipulate and remain flexible even at high gel percentages. This agarose offers a higher resolution of small fragments than the Certified molecular biology agarose.
Influence of gel percentage on optimal fragment resolution
Separations of Very Small FragmentsFor the separation of extremely small PCR fragments and primers (~10 to 200 bp), Certified low range ultra agarose is recommended. This agarose provides exceptionally higher resolution than standard gels at lower concentrations, allowing visualization of differences of ~5 bp in the 10–100 bp range. In this low range, the low range ultra agarose provides higher resolution than the PCR agarose.
Low-Melt Applications for Fragments <1000 bpFor applications such as cloning, in-gel applications, or DNA and RNA fragment recovery, Certified PCR low-melt agarose is recommended. This agarose has high-sieving capacity and offers excellent resolution of fragments <1000 bp in low-melt applications.
Low-Melt Applications for Fragments >1000 bp For separations of molecules greater than 1000 bp for clamped homogeneous electrical field (CHEF) separations, recovery of a larger fragment, or sealing IPG strips in 2-D SDS-PAGE gels, Certified low-melt agarose is recommended.
Separation of Large DNA for Pulsed Field Gel Electrophoresis (PFGE)For analytical separation of large DNA fragments requiring PFGE, pulsed field Certified agarose is recommended and an optimal separation range of 1 kb to 2 Mb is available as a preset, selectable method of the CHEF Mapper® XA system. This agarose can also be used for blotting.
Faster Migration Rate for CHEF and FIGEFor CHEF applications, PFGE, separation of megabase DNA, or other large fragment analysis, Certified megabase agarose is suggested due to its high exclusion limit, high electrophoretic mobility, and very high gel strength. The gels are easy to handle even at 0.3%, offer short run times, and have a separation range between 1 kb and 5 Mb.
Certified megabase agarose has superior electrophoretic mobility, faster migration rates, faster running times, and better separation of megabase fragments than the pulsed field Certified agarose.
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