Friday, July 2, 2010

New Gels, New Challenges

Note: I realize these posts may be confusing to those outside of biology/biochemistry fields. In general, gel electrophoresis separates molecules by size: In this case, biggest are at the top and smallest are at the bottom. For more, look at this great animated introduction.

As you remember from my last post, our last gel ran quite well, but the results showed that we had failed to digest our inter-nucleosome DNA. This time, we modified our digestion process (and by modify, I mean went back to the process I had been using before I decided to "improve" my process).

This time however, we have another issue. The gel itself gave us problems. There were a couple issues layered on top of one another, but the main one was that our power supply that runs the gel decided to quit some time in the middle of the night. This had two effects, one was that the DNA had time to diffuse and migrate in any old direction it wanted to during the time that the power supply was off. This causes a broadening of the bands which I think we are seeing here. Another issue was that it forced us, due to time considerations, to ramp up the voltage to very high values which I think is what caused the intense upside-down V-like shapes to the bottom of our gels. Finally, we ran the gel a little too long losing our 100bp marker. In the previous post, you could see the 100bp and 330bp markers, but I am pretty sure that the marker you see in the first lane is simply the 330bp marker. This makes it impossible to determine as, even though some of the middle markers are resolved, we have no way of counting up from the markers (each sub marker is 10bp).

As for the digestion itself, things look pretty good. We got rid of our smear of DNA and it is obvious that the DNA is migrating to approximately the correct place. There are only two issues I am worried about. One is that aside from the zero time point (the second lane), most of the lanes look the same. In other words, it seems as if we are completely digested by 5 minutes.

The second issue is that the lanes that do look different are completely random. The corresponding times are 20, 40, and 50 minutes. By 50 minutes, we shouldn't see anything above that band of brightness on the bottom. The fact that it is random seems to indicate a possible pipetting error. However, I ran and loaded two separate gels, so the pipetting error would have had to have come earlier. We'll try going over our process to make sure that this isn't an issue.

Other than that, it's back to do the exact same thing again. This time we'll be using a different power supply so we don't run into the same problems.

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