When we last left our DNA sample, it had been digested with a Restriction Enzyme at 37ºC for one hour. We are going to assume it has been digested at each site specific for the enzyme we used (although realistically that's far from always being the case). Is the DNA ready to load on the gel we poured? Not yet--there are several things we have to do first, but they don't take very long.

First, we need to stop the Restriction Digestion completely. Just because we remove the tube from the incubator does not mean the enzyme stops working. In fact, RE's will often cut DNA at random (nonspecific) sites at temperatures other than the optimum, like room temperature (which is about 23ºC unless you work on the 3rd floor of Burrill Hall, in which case it's around 40ºC). Have we discussed anything so far that you think might stop a Restriction Digestion? Yes, EDTA will stop the enzyme from working by denying it the divalent cations it needs. In fact, DNA is often stored in solutions containing small amounts of EDTA to prevent any stray nucleases from degrading the DNA.

[tube with loading dye] Next, what do you think would happen if we just loaded some of our sample into the wells of a gel and then filled the tank with buffer? The DNA sample, being in an aqueous solution (and therefore the same density as the buffer) would simply float away. Bad. What could we add to our sample to weigh it down so it remains in the well until current is applied? How about glycerol? Glycerol is indeed used, because it has a density greater than water. You may recall that glycerol inhibits RE's, but at this point, the digest is complete, the RE's have been inactivated with EDTA, and the glycerol will not harm the DNA fragments.

Lastly, we would like to monitor our DNA as it migrates across the gel. We can  do this--almost. Rather than mark the actual DNA, we can include charged molecules, called dyes, that are of two different sizes and colors. One is smaller than most or all of our DNA fragments, and will therefore run as fast or faster than the smallest DNA fragments. The other dye is large, and will migrate along with the very large DNA pieces. We can therefore assume that our sample is somewhere in between the dyes, so when the small dye gets near the end of the gel, the gel is stopped.

So when a digest is complete, the DNA sample is mixed with a combination of these three items, which appears blue due to the dyes. The tube Margaret is holding contains such a mixture, and it is called Loading Buffer, Tracking Buffer, or sometimes just Blue-Juice. Our sample is now ready to load. See, that didn't take so long!

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