Quick Guide: Agarose Electrophoresis
What is Electrophoresis?Electrophoresis is a technique that allows us to separate DNA, RNA or proteins according to their size.
What do I need to separate a mixture of DNA molecules?
In addition to your DNA sample, you will need:
- Gel Loading Solution – includes glycerol to help DNA samples enter into the wells and a visible dye to monitor migration through the gel.
- Agarose - a polysaccharide used as the separation matrix.
- Electrophoresis Buffer - contains ions necessary to conduct an electrical current, maintains pH of experiment.
- Horizontal Electrophoresis Buffer - holds the buffer and the gel, has positive and negative electrodes.
- Power Supply - generates the current necessary to move DNA through gel.
- Micropipette - used to transfer samples into wells.
- A special stain that allows us to visualize DNA.
How does electrophoresis separate DNA fragments?
The mixture of DNA molecules is added into depressions (or “wells”) within a gel, and then an electrical current is passed through the gel. Because the sugar-phosphate backbone of DNA has a strong negative charge, the current drives the DNA through the gel towards the positive electrode (Figure A).
At ﬁrst glance, an agarose gel appears to be a solid at room temperature. On the molecular level, the gel contains small channels through which the DNA can pass. Small DNA fragments move through these holes easily, but large DNA fragments have a more diﬃcult time squeezing through the tunnels. Because molecules with dissimilar sizes travel at diﬀerent speeds, they become separated and form discrete “bands” within the gel. After the current is stopped, the bands can be visualized using a stain that sticks to DNA (Figure B).
Casting the Agarose Gel
- 1. DILUTE concentrated 50X Electrophoresis buffer with distilled water (refer to Table A for correct volumes depending on the size of your gel casting tray).
- 2. MIX agarose powder with buffer solution in a 250 mL flask (refer to Table A).
- 3. DISSOLVE agarose powder by boiling the solution. MICROWAVE the solution on high for 1 minute. Carefully REMOVE the flask from the microwave and MIX by swirling the flask. Continue to HEAT the solution in 15-second bursts until the agarose is completely dis- solved (the solution should be clear like water).
- 4. COOL agarose to 60°C with careful swirling to promote even dissipation of heat.
- 5. While agarose is cooling, SEAL the ends of the gel-casting tray with the rubber end caps. PLACE the well template (comb) in the appropriate notch.
- 6. POUR the cooled agarose solution into the prepared gel-casting tray. The gel should thoroughly solidify within 20 minutes. The gel will stiffen and become less transparent as it solidifies.
- 7. REMOVE end caps and comb. Take particular care when removing the comb to prevent damage to the wells.
Running the Gel
- 8. PLACE the gel (still on the tray*) into the electrophoresis chamber. COVER the gel with 1X Electrophoresis Buffer (See Table B for recommended volumes). The gel should be completely submerged.
- 9. PUNCTURE the foil overlay of the QuickStrip™ with a pipet tip. LOAD the entire sample (35 μL) into the well in the order indicated by Table 1, at right.
- 10. PLACE safety cover on the unit. CHECK that the gel is properly oriented. Remember, the DNA samples will migrate toward the positive (red) electrode.
- 11. CONNECT leads to the power source and PERFORM electrophoresis (See Table C for time and voltage guidelines). Allow the tracking dye to migrate at least 3 cm from the wells.
- 12. After electrophoresis is complete, REMOVE the gel and casting tray from the electrophoresis chamber.
PROCEED to staining and visualizing agarose gels using FlashBlue™ Stain or SYBR® Safe DNA Stain.
*Gels that have previously been removed from their trays should be “anchored” back to the tray with a few drops of molten agarose before placing into the electrophoresis chamber. This will prevent the gels from sliding around in the trays and the chambers.