Gel Electrophoresis

Gel electrophoresis is a widely used technique for the analysis of nucleic acids and proteins. Most every molecular biology research laboratory routinely uses agarose gel electrophoresis for the preparation and analysis of DNA. We will be using agarose gel electrophoresis to determine the presence and size of PCR products.

Preparing the agarose gel 1%:

1. Measure 1g Agarose powder and add it to a 500ml flask

2. Add 100ml TAE Buffer to the flask

3. Melt the agarose in a microwave until the solution become clear (heat the solution for several short intervals - do not let the solution boil for long periods as it may boil out of the flask).

4. Let the solution cool to about 50-55°C, swirling the flask occasionally to cool evenly

5. Add BET with a final concentration of 200ng/µL

6. Seal the ends of the casting tray with two layers of tape

7. Place the combs in the gel casting tray.

8. Pour the melted agarose solution into the casting tray and let cool until it is solid (it should appear milky white).

9. Carefully pull out the combs and remove the tape

10. Place the gel in the electrophoresis chamber.

11. Add enough TAE Buffer so that there is about 2-3 mm of buffer over the gel.

Loading Samples and Running an Agarose Gel:

1. Add loading buffer 6X to each of your samples.

       Note: Loading buffer serves two purposes: 1) it provides a visible dye that helps with gel loading and will also allows you to gauge how far the gel has run while you are running your gel; and 2) it contains a high % glycerol, so after adding it your sample is heavier than water and will settle to the bottom of the gel well, instead of diffusing in the buffer. 

2. Carefully load a molecular weight ladder into the first lane of the gel.

       Note: When loading the sample in the well, maintain positive pressure on the sample to prevent bubbles or buffer from entering the tip. Place the very top of the tip of the pipette into the buffer just above the well. Very slowly and steadily, push the sample out and watch as the sample fills the well. After all of the sample is unloaded, push the pipettor to the second stop and carefully raising the pipette straight out of the buffer. 

4. Carefully load your samples into the additional wells of the gel.

5. Record the order each sample will be loaded on the gel, including who prepared the sample, the DNA template - what organism the DNA came from, controls and ladder

6. Run the gel usually at 100V during 30 mins or until the dye line is approximately 75-80% of the way down the gel.

7. Turn OFF power, disconnect the electrodes from the power source, and then carefully remove the gel from the gel box.

8. Using any device that has UV light, visualize your DNA fragments.