Pour agarose gels and analyze and compare DNA through electrophoresis based on size and charge
Genes, which are short DNA strands can be detected and compared through a DNA electrophoresis. DNA electrophoresis uses a high voltage DC power supply, like the Revolutionary Science RS-PS-75 and a DNA gel electrophoresis chamber, called the DNA RevBox. An agarose gel is made by mixing agarose powder and distilled water and boiling it in a microwave or Mini Pro Bath. When liquefied and homogeneous, the agarose can be poured into the casting tray, with a comb to create small wells where the DNA, mixed with electrophoresis dye can be inserted. When the gel and buffer solution and dyed DNA samples are added to the gel box, a DC current is applied using the power supply. As electrons move across the gel from the negative terminal (anode) to the positive terminal (cathode), the dyed DNA genes will advance across the gel based on size and charge. Genes that are smaller move faster across the gel, while genes that are positively charge will move in the opposite direction as the genes that are negatively charged.
Makes 200 mL. Use fresh or store jelled at room temperature (several weeks).
- Add 4.0 grams of agarose (electrophoresis grade) to 200 mL 1X TBE electrophoresis buffer in 500 mL beaker or flask.
- Stir to suspend agarose.
- Cover beaker with aluminum foil, and heat in boiling-water bath, like the Mini Pro Bath or in a Saniclave autoclave until all agarose is dissolved. This takes approximately 10 minutes.
- Swirl solution and check bottom of beaker or flask to insure that all agarose has dissolved (just prior to complete dissolution, particles of agarose appear as translucent grains). Reheat for several minutes if necessary.
- Cover with aluminum foil and lower the heat of the water bath to about 60C until ready for use. Remove any “skin” of solidified agarose from surface prior to pouring.
- Seal the ends of the gel casting tray with rubber stoppers or masking tape for the RevBox and insert a well forming comb.
- Pour the 2% agarose solution into the tray to a depth that covers about one third of the height of the comb teeth.
- Allow the agarose gel to completely solidify; this takes approximately 20 minutes.
- Place the gel into the electrophoresis chamber and add enough 1X TBE buffer to cover the surface of the gel.
- Carefully remove the comb and add additional 1X TBE buffer to fill the wells and just cover the gel, creating a smooth buffer surface.
- Orient the gel so that the wells are along the top, near the anode (black) side of the RevBox. Use a micropipet with a fresh tip to load 20 uL of pBR322/BstNI size marker into the far left well.
- Use a micropipet with a fresh tip to load 5 uL of each sample into your assigned well.
- Store the remaining 20 uL of your PCR product on ice or at -20C until you are ready to submit your samples for sequencing.
- Run the gel for approximately 30 minutes at 150V. Adequate separation will have occurred when the cresol red dye front has moved at least 50 mm from the wells.
- View the gel using a UV, blue light or white light transilluminator, depending on the stain used. Photograph the cells using a digital camera for later analysis.
Copyright 2014, DNA Learning Center, Cold Spring Harbor Laboratory. All rights reserved.