Analysis of nucleic acids by gel electrophoresis

/Analysis of nucleic acids by gel electrophoresis
Analysis of nucleic acids by gel electrophoresis 2018-02-23T10:35:24+00:00

Analysis of nucleic acids by gel electrophoresis

Analysis of nucleic acids by gel electrophoresis

Introduction

The term electrophoresis describes the migration of charged particles under the influence of an electrical field. Many biological molecules such as amino acids, proteins, peptides, nucleotides and nucleic acid possess ionisable groups and therefore at any given pH exist in solution as electrically charged species either as cation or as anion. Under the influence of an electrical field these charged particles will migrate either to cathode or to anode depending on the nature of their net charges.

The equipment required for electrophoresis consists basically of two items a power pack and an electrophoresis unit. The power pack supplies a direct current between the electrodes in the electrophoresis unit. All electrophoresis is carried out in an appropriate buffer which is essential to maintain a constant state of ionization of the molecules being separated. When potential difference is applied across the electrodes it generates a potential gradient which is the applied voltage divided by the distance between the electrodes.

The factors affecting the rate of DNA migration in agarose gels are molecular size of DNA, agarose concentration, conformation of DNA, applied voltage, direction of electric field, presence of intercalating dyes and composition of the electrophoresis buffer.

Requirements

Reagents

  1. Agarose
  2. DNA staining solution
  3. Electrophoresis buffer
  4. 6x Gel-loading buffer
  5. DNA samples
  6. DNA size standards

Composition of reagents and buffers

6x Gel-loading Buffer 

  1. 0.25% (w/v) bromophenol blue
  2. 0.25% (w/v) xylene cyanol FF
  3. 40% (w/v) sucrose in H2O
  4. Store at 4°C.

DNA Staining Solution

  1. Ethidium bromide (10 mg/ml)

Electrophoresis buffer

TAE

  1. Prepare a 50x stock solution in 1 liter of H2O
  2. 242 g of Tris base
  3. 57.1 ml of glacial acetic acid
  4. 100 ml of 0.5 M EDTA (pH 8.0)

Equipments

  1. Horizontal electrophoresis buffer
  2. Power supply
Agarose conc. in gel [% (W/v)] Range of separation of linear DNA molecules (kb)
0.3 5 – 60
0.6 1 – 20
0.7 0.8 – 10
0.9 0.5 – 7
1.2 0.4 – 6
1.5 0.2 – 3
2.0 0.1 – 2

Procedure

  1. Seal the edges of a clean, dry glass plate (or the open ends of the plastic tray supplied with the electrophoresis apparatus) with tape to form a mold. Set the mold on a horizontal section of the bench.
  2. Prepare sufficient electrophoresis buffer (usually 1x TAE or 0.5x TBE) to fill the electrophoresis tank and to cast the gel.
  3. Prepare a solution of agarose in electrophoresis buffer at a concentration appropriate for separating the particular size fragments expected in the DNA sample(s): Add the correct amount of powdered agarose (please see table below) to a measured quantity of electrophoresis buffer in an Erlenmeyer flask or a glass bottle.
    Range of Separation in Cells Containing Different Amounts of Standard Low-EEO Agarose
  4. Loosely plug the neck of the Erlenmeyer flask with Kimwipes. If using a glass bottle, make certain the cap is loose. Heat the slurry in a boiling-water bath or a microwave oven until the agarose dissolves.
  5. Use insulated gloves or tongs to transfer the flask/bottle into a water bath at 55°C. When the molten gel has cooled, add ethidium bromide to a final concentration of 0.5 μg/ml. Mix the gel solution thoroughly by gentle swirling.
  6. While the agarose solution is cooling, choose an appropriate comb for forming the sample slots in the gel. Position the comb 0.5-1.0 mm above the plate so that a complete well is formed when the agarose is added to the mold.
  7. Pour the warm agarose solution into the mold.
    The gel should be between 3 mm and 5 mm thick. Check that no air bubbles are under or between the teeth of the comb. Air bubbles present in the molten gel can be removed easily by poking them with the corner of a Kimwipe.
  8. Allow the gel to set completely (30-45 minutes at room temperature), then pour a small amount of electrophoresis buffer on the top of the gel, and carefully remove the comb. Pour off the electrophoresis buffer and carefully remove the tape. Mount the gel in the electrophoresis tank.
  9. Add just enough electrophoresis buffer to cover the gel to a depth of approx. 1 mm.
  10. Mix the samples of DNA with 0.20 volume of the desired 6x gel-loading buffer.
  11. Slowly load the sample mixture into the slots of the submerged gel using a disposable micropipette, an automatic micropipettor, or a drawn-out Pasteur pipette or glass capillary tube. Load size standards into slots on both the right and left sides of the gel.
  12. Close the lid of the gel tank and attach the electrical leads so that the DNA will migrate toward the positive anode (red lead). Apply a voltage of 1-5 V/cm (measured as the distance between the positive and negative electrodes). If the leads have been attached correctly, bubbles should be generated at the anode and cathode (due to electrolysis), and within a few minutes, the bromophenol blue should migrate from the wells into the body of the gel. Run the gel until the bromophenol blue and xylene cyanol FF have migrated an appropriate distance through the gel.
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