Monday, May 11, 2009

Agarose Gel Electrophoresis of Nucleic Acids

After amplify DNA template using PCR method, now you can continue your work by using gel electrophoresis in a gel composed of agarose in order to separate DNA fragments based on its molecular weight. The percentage of agarose used depends on the size of fragments to be resolved. In general a 0.8-1% gel may be used for effective separation of DNA fragments of 100-1500 base pairs.

Here are materials that you need:
  1. Agarose (molecular biology grade).
  2. Running buffer. There are two common types of buffer systems used in agarose gel electrophoresis, Tris-borate EDTA (TBE) and Tris-acetate EDTA (TAE).

    To prepare stock solutions:

    10x TBE buffer: 545 g Tris, 278 g boric acid, 46.5 g EDTA in 5 L of sterile distilled water.

    50x TAE buffer: 242 g Tris, 57.1 mL glacial acetic acid, 100 mL 0.5M EDTA, pH 8.0, in 1 L of sterile distilled water.
  3. TAE gel buffer systems are preferred to TBE systems when post-separation methods such as extraction with solid matrices are to be used. However, there is little difference between the two systems for general-purpose separation of DNA.
  4. Sterile distilled water.
  5. Microwave oven and boiling water bath or steamer.
  6. Loading buffer 50% (v/v) glycerol, 50 ruM EDTA, pH 8.0, 0.125% (w/v) bromophenol blue, 1.125% (w/v) xylene cyanol. Loading buffer is at 6X concentration.
  7. Molecular weight size marker: Numerous commercial DNA size markers are available, either as base pair ladders (e.g., 123 bp multimerladders) or predigested DNA (e.g., LambdaHindIII marker).
  8. Ethidium bromide: 10 mg/mL dissolved in H20. Store at 4oC in a container wrapped in tin foil.
  9. UV transilluminator (300 nm).
  10. Polaroid camera or gel documentation system.

And here is the method:
  1. The precise instructions for producing a gel depends on the gel-forming apparatus used. However, it is essential to make sure the gel casting stand is leak proof and sealed correctly (e.g., with end plates or waterproof tape).
  2. For a 1% agarose gel in TBE running buffer: Weigh an appropriate amount of powdered agarose into a conical flask. As an example, for a 1% agarose gel, add 1g of agarose and 10 mL 10X TBE running buffer and distilled water to the final volume of 100 mL, mix thoroughly by swirling.
  3. Heat the gel mix in a microwave oven (650 W) at full power for 1 min or less.
  4. Remove the gel mix and allow to cool to approximately 50oC or until just cool enough to hold.
  5. Pour the gel solution into a gel-forming tray, insert the comb template, and allow to set. This usually takes approx 25 min.
  6. Remove the comb and tape or end plates and add sufficient 1X running buffer to the tank to cover the gel and electrodes.
  7. To 5-10 mL of the DNA sample add 0.2 vol of loading buffer. Carefully add the sample to the well in one smooth pipetting motion.
  8. If the samples tend to float out of the well add an additional 1-2 vol gel loading buffer and reload the sample.
  9. Add an appropriate marker sample such as a Lambda-HindIII digest or a 123 bp ladder to the end wells of the gel.
  10. Carry out the electrophoresis at 100 V for approx 2 hours or until the bromophenol blue dye has travelled two-thirds of the way down the gel.
  11. Dismantle the gel apparatus and carefully place the gel in a tray. Add 100 mL of sterile distilled water containing 5 microliters of 10 mg/mL ethidium bromide (0.5 mg/mL) and allow to stain for 15 min.
  12. Destain the gel for 5 min by replacing the solution with fresh water.
  13. Discard the solution containing ethidium bromide in accordance with appropriate health and safety regulations. Remember to wear gloves when handling any solution containing ethidium bromide, which is a potent mutagen and potential carcinogen.
  14. Following electrophoresis remove the gel from the electrophoresis apparatus and view on a UV transilluminator.
  15. Photograph the gel using a polaroid camera or make a record using a gel documentation system.

Regards and happy electrophoresis.

Reference: Number 13 on References