Polyacrylamide gel electrophoresis (PAGE)

Vertical electrophoresis chamber, Glass plates, Casting holder, Pipettors, Gel imager

Gel electrophoresis is a method for separating biological macromolecules (such as nucleic acids or proteins) by forcing them through a gel matrix under an electric field. Because of the different charges associated with amino acid side chains, PAGE can be used to separate intact proteins based on net charge and size. The convergent motion of charged material in an electric field is called electrophoresis. Nucleic acid electrophoresis is usually performed in agarose gels or polyacrylamide gels. Agarose and polyacrylamide in different concentrations can form gels with different molecular sieve mesh sizes, which can be used to separate nucleic acid fragments of different molecular weights.

MCE has not independently verified the accuracy of these methods. They are for reference only.

1. Clean the glass plates and spacers thoroughly. Hold the plates by the edges or wear gloves, so that oils from the hands do not become deposited on the working surfaces of the plates. Rinse the plates with deionized water and ethanol and set them aside to dry.

2. Assemble the glass plates with spacers in gel caster.

3. Prepare the gel solution with the desired polyacrylamide percentage according to the table below, which gives the amount of each component required to make 12 mL:

4. Work quickly after addition of TEMED to pipet the acrylamide solution between the casting plates using a 5 mL pipetor before the acrylamide polymerizes.

5. Immediately insert the appropriate comb into the gel, being careful not to allow air bubbles to become trapped under the teeth. The tops of the teeth should be slightly higher than the top of the glass. If necessary, use the remaining acrylamide gel solution to fill the gel mold completely. Make sure that no acrylamide solution is leaking from the gel mold.

6. Allow the acrylamide to polymerize for 30-60 minutes at room temperature.

7. After polymerization is complete, surround the comb and the top of the gel with paper towels that have been soaked in 1x TBE. Then seal the entire gel in Saran Wrap or plastic bag and store it at 4°C until needed.

8. When ready to proceed with electrophoresis, remove gels from gel caster, carefully clean spilled gel from back of white plates and insert gels into gelbox. Add running buffer and carefully pull the combs from the polymerized gel.

9. Use a syringe to flush out the wells once more with 1x TBE. Mix the DNA samples with 6X loading buffer, load the mixture into the wells.

10. Connect the electrodes to a power pack, turn on the power, and begin the electrophoresis run.

11. Run the gel until the marker dyes have migrated the desired distance. Turn off the electric power, disconnect the leads, and discard the electrophoresis buffer from the reservoirs.

12. Detach the glass plates. Lay the glass plates on the bench. Use a spacer or plastic wedge to lift a corner of the upper glass plate. Check that the gel remains attached to the lower plate. Pull the upper plate smoothly away. Remove the spacers.

13. Stain gels with SYBR Green (Dilute with TBE in a ratio of 3:10,000 or 4:10,000) and expose to Gel imager.

1. The glass plates must be free of grease spots to prevent air bubbles from forming in the gel.

2. It is important to use the same batch of electrophoresis buffer in both of the reservoirs and in the gel. Small differences in ionic strength or pH produce buffer fronts that can greatly distort the migration of DNA.

3. Do not attempt to expel all of the sample from the loading device, as this almost always produces air bubbles that blow the sample out of the well. However, it is important not to take too long to complete loading the gel; otherwise, the samples will diffuse from the wells.

4. Nondenaturing polyacrylamide gels are usually run at voltages between 1 V/cm and 8 V/cm. If electrophoresis is carried out at a higher voltage, differential heating in the center of the gel may cause bowing of the DNA bands or even melting of the strands of small DNA fragments.