Gel Electrophoresis

Purpose

A PCR reaction is usually followed by gel electrophoresis. Gel electrophoresis is a technique used to separate different lengths of DNA, usually to determine sequence or to isolate a desired segment. In the Moore lab, gel electrophoresis is almost always used with the intent of determining whether the sample contains the correct segment of DNA, and to separate it. For example, a segment of vimentin will be A base pairs (bp), a segment of neon green will be B bp, and the segment containing both genes will be A+B bp. To obtain a sample of A+B segments, it would need to be separated via gel electrophoresis.

Preparation

To prepare for gel electrophoresis, the sample of DNA is generally amplified via PCR. While the thermocycler is running, a gel is prepared. To make a small gel, the gel will contain 50 ml of 1X Tris base + acetic acid + EDTA (TAE), and 0.5 g of agarose powder. The agarose powder is massed in a weigh boat and poured into the flask of TAE. Because the agarose powder will not dissolve on its own at room temperature, the mixture is microwaved for about 45 seconds. Once the agarose powder has dissolved, 5 μl of gel red is added with a 20 μl pipette. Gel red is responsible for flourecing when DNA is present which makes it possible to take UV photos of the DNA bands once the process is done. This gel solution is then poured into a gel mold which consists of the primary rectangular shape and a comb which will form the well indentations at the start of the gel (see figure 1). This gel solidifies for about 30 minutes before the comb is removed.

Gel Loading

After the DNA has been amplified, a blue-tinted loading dye (from Galadriel the -20॰ freezer) is added to the DNA sample so that bands can be monitored for completion. About 10 μl of this solution is then aliquoted into all but one of the wells. The first well is saved for the ladder. The ladder is a collection of known lengths of DNA segments which will provide a sort of ruler to compare your results to (see figure 2). The gel is then placed into the electrophoresis chamber and submerged with TAE. A cover is put on the chamber and connected to the electrodes, the negative one on the DNA side, and a positive one on the opposite side (see figure 3). A current then runs through the chamber for about an hour as the negatively charged DNA moves toward the positive end of the gel. Bands of DNA are separated during this process according to size because smaller segments of DNA can more easily move through the gel and therefore move farther than the larger segments.

Gel Imaging

After the DNA has moved a reasonable distance (varies depending on length of sample segments and concentration of gel) the gel is carried to the UV camera. Because UV light is largely damaging, it is important to limit the time that the DNA is exposed to the UV rays. This is why the light is turned on, the image is captured, and the light is immediately turned off. The computer program also allows the user to edit the brightness and invert the colors to make the bands as clear as possible (see figure 4). We then usually print out a few copies for our lab notebooks and compare them to our ladder key to determine if the desired DNA segments are present. If they are and need to be harvested, the next step would be gel extraction.