Here we are! The fruits of our labor are now before us. This photo shows the polaroid photograph of the gel we ran. Actually, it's not the gel we ran in this experiment, but it shows what we are interested in more clearly.
The white bands represent DNA of a particular size. The arrows are included to point out bands that are legitimate, yet might be overlooked as background noise until you have looked at enough gels to recognize them. Remember what we discussed earlier about Ethidium Bromide intercalation? Can you think of two reasons one band is brighter than another?
Lanes 1-3 contain our DNA samples, perhaps from the same stock of DNA digested with 3 different Restriction Enzymes, or maybe from different pieces of DNA altogether. When we are interested in determining the success of a particular digest, we often want to calculate the size of these bands. How can we do this? We use the information in lanes 4 and 5, and a little math.
Lanes 4 and 5 are called Standard Lanes, or sometimes Molecular Weight Markers or "Ladders". They are similar to the Control in an experiment, because we know exactly how they are going to turn out every time. These lanes contain DNA of a specific, predetermined size from a known source, digested with a Restriction Enzyme that cuts that piece of DNA a known number of times, yielding a predicted number of bands whose size we know exactly. For example, Lane 5 is DNA from the E. coli bacteriophage Lambda, digested with Hin dIII. We know exactly how long each of the visible fragments are.
There is a logarithmic relationship between the size of a DNA fragment and the distance it migrates on a gel, so by measuring how far the bands in our various standard lanes (4 and 5 in this case) migrate, we can construct a standard curve since we know the fragment sizes. Then we measure the distance our experimental bands migrated (lanes 1-3), and plot it on our standard curve. This will give us the approximate size of the experimental fragment. The more points on the standard curve, and the more separation we get on our gel, the more accurate our approximation will be.
One important sample every experiment should include is uncut DNA. Uncut DNA exists in as many as three different forms: supercoiled, relaxed circular, and linear. Just because you didn't add any RE to your sample does not mean it hasn't gotten nicked or even cut at some point in its preparation. Nicking causes supercoiling to be relaxed, while cutting as you know causes linearization. Nothing has been removed from this piece of DNA, so all 3 forms are the same size. Does that mean they will migrate the same? Unfortunately no. Imagine a rubber band all twisted up into a little knot. That is very much like the compact form of supercoiled DNA. The relaxed, unknotted rubber band is similar to the circular form, and if you were to cut the rubber band once with scissors, it would resemble linear DNA. Which will experience the most resistence in an agarose gel? The least? From fastest to slowest is supercoiled, circular, then linear, despite the fact that all three fragments are of exactly the same length. In the photo above, Lane 2 is most likely to be the uncut sample. That observation is based solely on experience in looking at gels--you would need to know exactly which sample went into each lane to be certain.
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