Gel Electrophoresis

Gel electrophoresis is a method utilized to visualize DNA and is how we will determine if the PCR reactions yielded the correct results. DNA is pushed through a gel matrix by an electrical current. Because DNA is negatively charged, the positive current will cause the DNA to migrate down the gel toward the positive electrode, separating fragments into discrete bands. These DNA bands are visualized by adding a stain activated by UV light and compared using a DNA ladder, which is composed of bands of a known size for comparison.

Prepare the gel:

1) Measure out the appropriate amount of agarose to create the desired density gel. Add the agarose to a flask.

*in 100mL total, 1g agarose = 1% gel

2) Add the appropriate amount of 1x TAE buffer to create the desired density gel.

3) Heat the bottle in the microwave in 1-minute increments. Watch the flask to ensure the agarose does not boil over. Occasionally remove the bottle and swirl it gently to distribute the heat.

*Be careful, as the gel heats it will produce a lot of steam!

4) Allow the agarose to cool until it can be handled comfortably with your bare hand. Do not allow it to over-cool, as it will begin to solidify.

5) Add SYBR Safe dye to the melted gel and swirl it to mix. The gel will be a slightly pink color. Pour it into the gel box.

*1uL of SYBR Safe for every 10mL of gel

6) Allow the gel to completely solidify. It will be slightly opaque and will not move when you tap the gel box.

7) Remove the comb and turn the tray in the gel box. The wells should be toward the black electrode.

8) Add enough 1xTAE buffer to completely cover the surface of the gel.

Running the gel:

1) Prepare the DNA samples

2μL of 6x loading dye (blue dye) to 5μL of the DNA sample

2) Carefully load the ladder into the gel. Use 7μL of the ladder.

*Use the 1Kb ladder for checking DNA extractions (labeled “1%”)*

*Use the low range ladder for checking PCR (labeled “2%”)*

3) Load the DNA samples into the well of the gel. Be careful not to puncture the bottom of the well and avoid bubbles, as this will cause the sample to float out of the well.

4) Plug in the power supply and turn on the gel. Run the at 170V until the dye front has reached the desired position on the gel.

*Position of the dye front is more important than time. It may take more or less time to get your gel to the right position, so check it regularly.

5) Visualize the gel using a UV transilluminator and take a picture of the gel.

Blue dye runs ~here

Yellow dye runs ~here