Molecular Biology. A Practical Guide - Velikov V.A. 2013

Nucleic Acid Electrophoresis
DNA Gel Electrophoresis

Gel loading buffer. DNA samples are loaded into the gel wells using a buffer containing glycerol or sucrose. This ensures that the DNA immediately settles to the bottom of the well rather than dissolving into the Electrophoresis buffer before the current is applied and the DNA enters the gel. Special tracking Dyes are added to the buffer to monitor the progress of the electrophoresis front. The electrophoretic mobility of bromophenol blue (BPB) corresponds to approximately 100 Base Pairs. The BPB blue band migrates alongside tRNA as a front-running dye. The mobility of xylene cyanol (XC, green) corresponds to approximately 460 NUCLEOTIDES.

DNA quantity per gel lane. The lower detection limit for DNA visualization depends on the method used. When EtBr staining is applied, as little as 10 ng of DNA can be detected in a 5 mm wide band. An excessive amount of DNA per lane (“overload”, >1 µg) alters band mobility, causing it to migrate faster, and leads to band smearing. The optimal amount of DNA to load into a well depends on the number and size of the DNA fragments, typically ranging from 0.2 to 0.5 µg per well.

Genomic DNA, unlike plasmid and phage DNA, invariably produces a smear of varying-sized fragments. Under UV light, uniform staining is observed across the entire length of the gel, commonly referred to as a “smear” (from the English term). Lane 3 in App. 13 demonstrates this type of DNA smear.

Electrophoresis buffers. Buffers containing 50 mM Tris-acetate, Tris-borate, or Tris-phosphate at pH 7.5–8.0 are commonly used. They are usually prepared as concentrated stocks and stored at room Temperature. Tris-borate and Tris-phosphate buffers have a higher buffering capacity and provide better resolution; however, if subsequent DNA Hybridization or sequencing is required, Tris-acetate buffer is preferred.

Field strength. Effective Separation of DNA fragments is achieved at an electric field strength not exceeding 5 V/cm of gel. Higher field strengths impair separation efficiency. Elevated temperatures cause DNA molecules to readily lose EtBr, which frequently occurs during high-voltage electrophoresis. Ethidium bromide migrates from “+” to “−” during electrophoresis, i.e., in the direction opposite to DNA migration. To prevent it from migrating out of the gel—for instance, when low amounts of DNA are loaded—it should be added directly to the buffer.

Mobility of different DNA Conformations. Double-stranded DNA molecules with identical molecular weights but different conformations migrate at varying speeds. For example, the supercoiled circular, nicked circular (open circular), and Linear Forms of a plasmid migrate through an agarose gel at different rates (App. 9).

In a 1% agarose gel, single-stranded DNA (and RNA) migrates approximately 10% faster than double-stranded DNA of the same size. This DNA (RNA) stains approximately 4–5 times less intensely with ethidium bromide, a factor that must be taken into account when visually evaluating electrophoresis results.

Materials and Equipment

Agarose gel, bacterial, plant, or animal DNA solutions obtained from experiments 1.1–1.3, molecular weight marker (commercial preparation) or HindIII-digested phage λ DNA, power supply, electrophoresis chamber, UV transilluminator.

Solutions

- 10× loading buffer: 0.025 g bromophenol blue (BPB); 4 mL glycerol; 5 mL 0.5 M EDTA, pH 8.0; bring to 10 mL with double-distilled Water.

Procedure

1. Place the gel tray into the electrophoresis chamber and pour in 1× TBE buffer (experiment 3.1) so that it completely covers the agarose ^a.

2. Mix 10 µL of DNA solution and 1 µL of 10× gel loading buffer in a microcentrifuge tube, a well of a small immunological plate, or simply On the surface of a Teflon comb. Mix thoroughly by pipetting.

3. Load the sample(s) into the gel well using a micropipette.

4. Load the molecular weight marker into an adjacent gel well.

5. Turn on the power supply and perform electrophoresis at 100 V for 1.5–2 hours (until the tracking dye BPB migrates out of the gel).

6. Turn off the current, remove the tray, and visualize the gel under UV light.

7. Photograph the gel using a digital camera ^b (for photograph, see App. 13).

Notes

^a The buffer layer above the agarose should be up to 5 mm deep; otherwise, the gel may dry out.

^b Unlike polyacrylamide gels used for Proteins, agarose gels containing DNA should be photographed immediately, as DNA bands tend to diffuse and blur during storage. Experience shows that by the following morning, the bands lose their definition.