DNA Electrophoresis in Agarose Gel

Materials

• Tris-Borate Buffer(10X; pH8.2)
  0.9M Tris-HCl113.0g
  0.025M EDTA Na₂ 9.3g
  0.9M boric acid55g
  Double distilled water1L
• Agarose. 1.0 (wlv) in single-strength Tris-borate (gel-running) buffer.
  Autoclave to dissolve the agarose; then maintain at 50°C in a stoppered flask until used.
• Gel-loading Solution:
  Sucrose 30%, Bromophenol blue 0.25% and Xylene cyanole FF 0.25% (all wlv) in single-strength Tris-borate buffer.
• DNA Preparations (Plant DNA, plasmid DNA, recombinant DNA etc.)
• Standard DNA for Size Determination
  (l DNA cut with restriction endonucleases Eco RI and Hind III)
• Gel Casting Plate
  Gel tank, power pack etc.
• UV transilluminator with photography system, safety glasses.

1. Form a wall around clean dry, gel-casting glass plate (10 X 6 x 0.5cm) using zinc oxide tape. This should give a leakproof wall about 1cm high all around the plate. Alternatively, the plate is placed in a suitable gel casting tray purchased from a commercial supplier. Place the set-up perfectly horizontal over a leveled plate.
2. Pour 30mL of 1% agarose solution maintained at 50°C onto the casting plate. Immediately place a suitable well-forming comb about 1cm from one end of the plate.  The teeth of comb should not touch the glass plate. Allow the gel to set for 1h.
3. Remove the comb from the gel, carefully. Transfer the gel along with the glass plate to the electrophoresis tank such that the wells are near the cathode. Pour single-strength Tris-borate buffer into the tank until the gel (including zinc oxide wall) is submerged.
4. Connect the electrodes to the power supply with the cathode (-ve) at the well end of the gel.
5. Load DNA samples (5-20//L) and standard taken in gel-loading solution using a microsyringe.
6. Turn on the power supply and run at 100V (10-15mA). Monitor the progress of fast-running (bromophenol blue) tracking dye during electrophoresis. Terminate the run when the tracking dye is about to leave the gel.
7. After disconnecting the power supply, transfer the gel to a staining tray containing 250/ig ethidium bromide (50//L of 5mg/mL solution) in 250mL of used Tris-borate buffer. Stain the gel for about 30 min.
8. Transfer the gel onto a thick plastic sheet, place on a UV transilluminator and view the gel under ultraviolet light (300nm). Nucleic acids on the gel will appear orange owing to the fluorescence of bound ethidium bromide. Photograph as soon as the gel has been checked for the presence of bands. Use the photograph for further interpretation of band patterns.
9. Measure (from photograph) the distance moved by each band from the front of the loading well. Plot the distances against log molecular weight of standards (A DNA fragments) to give a calibration curve. Deduce the size of DNA and/or restricted fragments of samples using the curve.

Notes

1. Agarose used for electrophoresis should be of very high quality - low sulphate content and electroendosmosis (e.g., Sigma Type I grade).
2. Agarose gels of different thickness, concentration (0.2-2%) and dimensions are used depending upon the experimental requirement.
3. Pour the gel carefully without entrapping any air bubbles. Keep the agarose solution always at 50-55°C until poured on the casting plate to avoid setting. Handle the gel carefully as it is delicate.
4. Minigels are run at high current for quick results. Otherwise, the gels of large size are usually run at a constant low voltage (25-40) overnight for high resolution of bands.
5. Wear gloves while using ethidium bromide as it is mutagenic.
6. Ethidium bromide may be added to the gel running buffer itself. The separation of bands then can be followed even during electrophoresis using a hand-held UV lamp.
7. Wear safety glasses for viewing the gel under UV light.   In order to prolong the life of the transilluminator and to avoid excessive exposure to UV radiation, photograph the gel at the shortest period and use the photograph for further interpretation. Position a mini-scale and photograph to measure the relative mobilities of bands later.
8. The electrophoretic mobility of bands is very much affected by the salt concentration in the sample. It is important that the salt concentration in samples loaded in various wells is (nearly) uniform.
9. The sizes of restriction fragments from lambda DNA (49kb) are given below:

References