Your gel should look something like this, I’ve drawn this one with the wells at the bottom of the picture. In this example gel, I’ve run a standard and 4 samples. The standard is essential to analyzing this data. Each gel is individually poured and the electric field in the electrophoresis unit might not be absolutely uniform, so the standard lane is included to permit interpretation of the data. Let’s take a look at that standard… Std #1 #2 #3 #4
The size of the fragments in the standard must be given so a relationship between fragment size and migration distance can be determined. The “size” of a DNA fragment can be reported as the number of DNA base pairs in the fragment. Since these agarose gels act like a sieve, the smaller a DNA fragment is, the farther it can migrate through the tangled mess of agarose. For this standard, let’s say that the fragment sizes are: 12,000bp; 4000bp; 900bp and 300bp. To measure the migration, we need to actually measure the distance each spot travelled… Std #1 #2 #3 #4
Note, I don’t have an unit specified on the “ruler” here, because the units really don’t matter as long as you are using a consistent measuring unit for all of your spots. One of my professors when I was an undergraduate used to use the teeth of a comb to measure things like this and it worked just as well as the fanciest ruler you can find. If we want to calculate a proper retention factor (Rf) for these spots, we also need to know the distance the parker dye travelled. On your gels, the marker dye washed away during destaining, so we can estimate where the dye front was, it’s somewhere above your highest spot. Don’t make “Rf” into some magical new concept, it’s really just the fractional distance that the spots migrate. If you drive on the highway for 3 hours, your possible distance travelled is 3x70=210miles. If you had a bathroom break in there, you might have only travelled 185miles during that 3 hours, so your “Rf” for the trip is 185/210=0.88. Chromatography/migration works exactly the same way. dye front, 53 units 300 bp 900 bp 42 units 4,000 bp 32 units 23 units 12,000 bp 13 units Although calculating Rf is considered proper analysis of these gels, you could get perfectly usable results if you just use the migration distance directly. Std #1 #2 #3 #4
Organizing the data from the previous slide… Now we want to figure out a mathematical/graphical relationship between the Rf of a spot and the number of base pairs in the DNA fragment responsible for that spot. That’s why I’ve included the “log(bp)” and “1/bp” columns in the table above. If we prepare graphs of base pair vs. Rf, log(bp) vs. Rf, and 1/bp vs. Rf, we can look for a good relationship. The graphs are on the next slide. There’s a graph using Rf and a graph using migration distance, notice that they’re identical…
For the data that I made up for this example, it looks like the plot of log(bp) vs. (migration distance or Rf) gives an approximately linear plot. Is this true with real data? Let me emphasize, I made this data up off the top of my head, it may be an accurate reflection of real data or it could be absolute nonsense. Make sure you analyze your own data with all three graphs.
Measuring the unknown spots on the gel… dye front, 53 units 46 units, Rf=0.868 37 units, Rf=0.698 32 units, Rf=0.604 26 units, Rf=0.491 25 units, Rf=0.472 17 units, Rf=0.321 13 units, Rf=0.245 9 units, Rf=0.170 Std #1 #2 #3 #4
Using the fit equation from the linear graph, we can calculate the number of base pairs in each fragment based upon its Rf on the gel. None of these samples are from the same source, but you can use the calculated base pairs for your samples to determine if either of the suspects is the source of the DNA that was found at the crime scene.