Thursday, July 29, 2010

Many general lab updates

For our nucleosome project, we have had some setbacks, most of which we have recovered from. As you remember from last time, our column had broken. That is still the case and we are still waiting for a replacement as it is on back-order. What I had failed to mention was that the sample itself had also had some issues. Namely, there was a white powder that had precipitated out of the sample and to the bottom of the tube. This was disturbing as I was relatively sure that this was all of my nucleosomes aggregating and falling to the bottom of my tube (something they are only supposed to do when I want them to). Since we were stuck without column, I thought I would investigate this issue.

It turns out that nearly all of my nucleosomes had aggregated. After some investigation, it turns out that the nucleosome sample was actually in 10mM Calcium Chloride (a +2 ion) instead of 1mM. This is an issue because nucleosomes aggregate at about 5mM of +2 ions. It is also a problem because the EDTA we add to stop the digestion is added to stop 1mM of CaCl2, not 10mM.

The first thing I needed to do was get the nucleosomes back in solution. We did this by dialyzing the nucleosome solution (and the precipitate) with 150mM NaCl (a +1 ion). This is done by putting the nucleosome solution in a bag that only lets ions and water pass through it, but not the nucleosomes. It turns out, if you add enough +1 ions, they will displace the +2 ions at which point the nucleosome should go back into solution. After a day of dialysis, we had significantly increased the amount of nucleosomes in our solution. After a weekend of dialysis, we had gotten back basically all of our nucleosomes. Now we had to make sure that they had been digested correctly (especially checking for over-digestion which shows up as smearing on our gel).

The results look like this. Now the furthest column to the right is our DNA ladder. This is used to let us know how long our DNA is. You match the known length of the ladder band to your sample. The bright band at the bottom is a 100 base pair DNA. Each step "up the ladder" is a 10 base pair step (110 base pair, 120 base pair, etc). The lane immediately to the left of the ladder is our sample. If you look and count carefully, you can see that it lies between the 140 base pair and 150 base pair "steps" of the ladder. This is perfect as the DNA should be 146 base pairs! Even better, there is no "smearing" to lower parts of the gel, which would indicate over digestion. You can see an example of this in a different sample that was run in the lane immediately to the left of our sample.

So as a wrap-up, we have the same amount of nucleosome we started with, all in solution and digested to the correct length. All that is left to do is to separate out any small amounts of double or triple or larger nucleosome arrays and we will be done. Since we are still waiting for our column to arrive, we may end up doing this through a sucrose gradient instead.

Not to be shown up, John is busy plowing through the ton of data that he has taken. Now that we have all of the data, we need to figure out why some of it looks great and some of it looks not-so-great. Things looked pretty bad at the beginning of this process, but each time I check in with him, he seems to have found another correction to make the data fall in line. If all goes well, we'll have it all whipped into shape by the end of this week.

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