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Tutorial in Fast DNA Electrophoresis: Resolution of Larger DNA Fragments

Tutorial in Fast DNA Electrophoresis: Resolution of Larger DNA Fragments. Each conductive medium has its own “personality” that relates electrophoretic mobility and DNA fragment size.

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Tutorial in Fast DNA Electrophoresis: Resolution of Larger DNA Fragments

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  1. Tutorial in Fast DNA Electrophoresis:Resolution of Larger DNA Fragments

  2. Each conductive medium has its own “personality” that relates electrophoretic mobility and DNA fragment size. That is, some conductive media separate small DNA fragments well, and other media are favored for their separation of large DNA. It is difficult to find a straightforward explanation in a single place in the literature, and so this brief tutorial was prepared. Some of this discussion is found in our publication in Analytical Biochemistry 2004; 333:1-13.

  3. Four rules govern the optimal size-range of most conventional conductive media in continuous voltage gel electrophoresis. 1) Tris ions cause small DNA (50-500 bp) to form fuzzy bands. Acetate buffers also tend to produce fuzzy bands in the smaller size range. In contrast, borate ions allow sharp bands in all size ranges. 2) Borate is a bifunctional agent that complexes with DNA. It crosslinks DNA (probably both intramolecularly and intermolecularly) at low concentrations. This can cause the bands of large DNA fragments (over 3000 bp) to crowd. Borate at high (over 50 mM) concentrations saturates the DNA to reduce this crosslinking. 3) Acetate has an excellent size range, with superb separation of small and large DNA fragments (over 3000 bp). It seems unlikely that acetate has any special property in regards to large DNA. It is more likely that the mere absence of borate crosslinking is the key. 4) Large DNA is best seen when using lower contents of agarose in the gel. Standard agarose can be used as low as 0.7 or 0.8%. A high-strength agarose is available commercially that permits one to make agarose gels as low as 0.3%. In a 0.3% gel, good separation of large DNA is achievable in even borate buffers.

  4. 300V 400V 0.3% 2% LB LB M M - 650 bp - 100 bp 8 9 Note that SB™ (lanes 1 and 2) and LB™ (lane 9) are excellent for small DNA but encounter crowding of the bands of larger DNA when run in standard agarose (lanes 1 and 5), possibly due to intramolecular DNA crosslinking, which reduces the discriminating shape differences among the larger molecules. TBE, having nine times the borate concentration of SB and LB, better saturates the DNA binding sites, aiding separation of larger DNA somewhat (lane 6), but at the expense of having a higher current. Its high current precludes fast DNA separation at higher voltages (lanes 3 and 4 show a failure of a TBE gel). The acetate in TAE provides an excellent separative pattern (lane 7), but, as with all tris media, cannot be run at high voltages. LB™ provides the best of all worlds, permitting a nice separation of large DNA, similar to TAE, when used with low-percent agarose gels, even under very rapid electrophoretic conditions (lane 8). Neither TAE nor TBE can produce the superb resolution of small DNA as can SB™ or LB™ (lane 9). A standard agarose was used at 0.7 to 2% (lanes 1-7 and 9) and a high-strength agarose was used at 0.3% (lane 8).

  5. LA™ Resolves Large DNA in Standard Agarose 300 V - 1.0% agarose When rapid electrophoresis of large DNA is desired but one wishes to use a standard agarose, LA™ is the choice. LA™ is excellent for larger DNA fragments when a strengthened low-percent agarose gel is not available. LA™ is lithium-based, borate- and amine-free. It can be run at higher voltage than TAE due to its low conductivity. Above is an un-retouched photograph of a 1 kb ladder from 3,000 to 12,000 bp run on a 1% standard agarose gel at 30V/cm. The gel migration pattern of LA™ is essentially identical to that of TAE, but the run was completed in a fraction of the time.

  6. LB™ Resolves Large DNA in High-Strength Agarose 300 V 0.3% agarose 25 min LB™ is excellent for larger DNA fragments when a high-strength low-percent agarose gel is available. LB™ is lithium- and borate-based and amine-free. It can be run at higher voltage than TAE due to its low conductivity. At right is an un-retouched photograph of a 1 kb ladder from 200 to 12,000 bp run on a 0.3% agarose gel at 30V/cm. The gel migration pattern of large DNA fragments in LB™ is de-compressed when run in 0.3% gels. The run was completed in a fraction of the time needed for a TBE or TAE gel. Because high-strength agarose is used in very low concentration, its cost per gel is identical to a standard agarose.

  7. The recommended approach: • Use SB™or LB™ for small and medium-sized DNA. This includes most PCR and RT-PCR products. Use high voltage (350-450 volts for a 10 cm gel, or 35-45 V/cm). 2% gels show the sub-500 bp region exceptionally well. Use a standard agarose. • Employ a high-strength agarose to permit use of 0.3 to 0.5% agarose to produce better separation of DNA fragments larger than 3000 bp fragments when using LB ™or SB ™. SeaKem® Gold (Cambrex) works well for this purpose. Use high voltage (30 V/cm). • Use LA™ for fragments larger than 3000 bp if using a standard agarose at 0.8 to 1.0%. LA™ takes advantage of a non-borate medium and the low electromobility of lithium ion. Use high voltage (30 V/cm). • For very large DNA (>10 kb), field-inversion techniques can be employed. One can use tris-based protocols as found in the prior literature, or use a low-conductance medium such as LB™, SB™, or LA ™ in combination with higher voltages.

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