Thursday, July 15, 2010

Great Successes and Amazing Failure

A lot has happened since I last posted. Let's start with the positive.

We have successfully run a very nice non-denaturing poly-acrylamide gel. The key was to increase the amount of sucrose in or loading solutions. This helped minimize diffusion of the samples before they started running. Another thing that helped was a mistake I made. In my haste to make the gel, I accidentally inserted a 10-well comb rather than a 15-well comb. However, this actually resulted in much nicer results.

We see exactly what we would like. The first column is our normal DNA ladder. The second is the undigested chromatin. As you can see, it is all at the top of the gel. Then we start digesting. As we go to the right, the time of digestion is increasing. As you can see, by the sixth column or so, the size doesn't change much for a few columns. This is the digestion being stopped right at the nucleosome core. After a bit of time, however, the digestion proceeds past this point in some of the nucleosomes, digesting the DNA that is around the core. That is why the gels get smeared towards the bottom for the last three columns. When we do our final digestion, we will make sure that we don't let it digest this long.
After this great success, we digested one of our samples and were going to pass it through our size-exclusion chromatography column. This column will separate our sample by size. This will allow us to collect all of the nucleosomes that are the exactly correct size, while ignoring those too big or anything that is too small (bits of DNA, etc.). The setup is shown to the right.

One thing about a size-exclusion column is that it must never dry out. The tricky thing is that you need it to almost dry out to add your sample so that your sample stays relatively concentrated in the gel. To make a long story short, I brought the column to the almost dry level and the closed the valve. However, the valve leaked slightly and after 15 minutes, when we loaded the sample, I noticed the gel had completely dried out.

At this point we needed to recover the sample (which we did right away). We then took the column back to the lab to recover the media (the white stuff in the column) so we could rehydrate and repack the column. During this process, I overtightened the column in the vise that was holding it. So now we are waiting on a replacement column and replacement media. It's funny how much a small leaky valve and 15 minutes can change things.

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