DNA Gel Extraction

Purpose

The purpose of a DNA gel extraction is to isolate DNA from a band that was separated via gel electrophoresis. During gel electrophoresis DNA is separated into bands depending on their size. Gel extraction allows the scientist to isolate the specific size band of DNA they want so that their aren't unwanted pieces of DNA in their sample. Gel extraction is a common procedure done in the Moore lab, and often something I get to do on my own.

Process

Gel extraction begins by cutting out a small segment of gel from the gel electrophoresis. To make the bands of DNA visible, the gel is placed on a small box that emits UV light. UV light causes the bands of DNA to fluoresce (see figure 1) because of the "gel red" that was added while pouring the gel (see "gel electrophoresis"). To prevent UV damage to the eyes, which can cause blindness, a face shield is worn, and there is also a shield built into the box which can be moved and opened like a door to allow you to work. The needed segment of DNA is removed with a razor and placed in a weigh boat so that it can be massed. For

The following procedure including all necessary solutions are included in one of the lab's gel extraction kits. There are kits for many types of reactions and they are especially handy because they contain complete and thorough instructions for you to follow. For experiments done on gels with concentrations less than or equal to 1%, a gel ratio of 1:3 solubilization buffer is added to a 1.5 mL micro-centrifuge tube along with the gel sample. This mixture is then incubated at 50॰C until the gel has completely dissolved. Once the gel has dissolved, up to 850 μl of the solution can be aloquotted into a wash column (see diagram). The column is spun in the centrifuge at 12,000 rotations per minute (rpm) for 1 minute so that the liquefied gel, solubilization buffer, and anything else unwanted can flow through the filter and be discarded. The DNA will be left atop the filter. After the flow-through is discarded, 500 μl of a wash buffer is added and the column is centrifuged again. After discarding that flow-through, the column is centrifuged dry at max speed for 2-3 minutes so that there is no wash buffer left on the DNA. This is important because the container of wash buffer that comes in the gel extraction kit contains 70% ethanol, and ethanol can corrode nucleotides if left for too long meaning the sample of DNA can be destroyed. Next, the sample is eluted either with elution buffer from the kit, or ultrapure H2O. For this step, the column is moved into a new collection tube that is clean and sterile so that the DNA is not harmed, and it is centrifuged so that the DNA can pass through the filter with the elution buffer or water and live safely at the bottom of the collection tube.

Staring at the tube of what appears to be a clear and colorless liquid, it is impossible to know what the DNA concentration is. Therefore, a mass spectrometer is used. The mass spectrometer is a cute little machine that can send waves through a small, 1μl drop of the sample and tells you the nucleic acid concentration, the 260/280 ratio, among other things. The nucleic acid concentration is merely the concentration of DNA that was collected. The 260/280 ratio is a ratio of nucleic acid count to random proteins and enzymes. It is called the 260/280 because nucleic acids have an absorbance maxima of 260, and proteins and other larger molecules have absorbance maxima of around 280. The 260/280 value is generally considered acceptable when it is in a range of 1.5-2.5.

Also...

When the column is placed in the final collection tube, there is the column cap which will be closed in the centrifuge, then the secondary outer cap on the collection tube will remain open while spinning. Something I learned is that during this final spin, the secondary caps will often fly off and shatter all over the inside of the centrifuge machine. This means that after the DNA is collected it almost always must be pipetted into a new centrifuge tube so that it can be closed and stored.