Today I ran 2 digestions with the only difference being how much nuclease i put in each sample. In one i put a 10x dilution of our nuclease and in the other i put a 100x dilution. I kept everything else the same and used our old protocols to complete the digestion. On Monday we are going to run the gel because we have been kicked out of the chemistry department for the rest of the week.
On Thursday, I made some new spreadsheets to analyze the data in a different way. I am trying to average the data in different ways, by averaging the values across the data series first. I think that doing this way is more accurate, because the trials are fairly consistent, and this helps to take into account where most of the error is coming from. This process seems to be making the data agree with itself more, which is promising.
On Wednesday, I finished running the concentrated trials of the second series. I also reprocessed a few of the earlier trials, trying different calibrations. I discovered and correct one major problem, which was that I had run out of sample in one of the trials. This was throwing that trial way off, because it was taking values when there was no sample left. Reprocessing some of the data with different calibrations also helped make the data more consistent.
On Tuesday, I continued to prepare and run the concentrated trials for the second series. So far things are going well, but the data is not quite matching up with the diluted trials. This could be because of the way I am analyzing the data, or also from problems and inconsistencies in the data itself.
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.
Today I ran tests on the Lambda 25 to see if it is properly working, and it passed all the tests I could do without having to make any samples. I also created an instruction sheet for the machine with the basics so that anyone can now use it.
Today I made and ran a second series of concentrated samples for the first series. The results came out somewhere in between the diluted series and the first concentrated series that I ran. This is promising, because the results should all be the same. It is possible that there is some sort of concentration dependent interference that is happening, which could be causing some of the differences. Today, I decided to start running concentrated trials of the second series, and started preparing the samples. This should be easier because there is much more of the second series left. Overall, I am finding that the eighth and tenth points are still uncharacteristically high, which leaves me to believe it is something in the actual sample preparation. This is one of the only explanations I can see left, because I have ruled out most of the possible problems on the testing end. One possible explanation is that not all of the Mg buffer solution was removed when the DNA precipitated.
Today, I wrote the second of two results reports, and updated all the graphs for the second series. It was Matt and Andrew’s last days, so we had their final presentations today as well.
On Thursday, I began by analyzing the concentrated trial for the first series. It came out to be significantly lower than the eight diluted trials I had run previously. By lower, I mean the the normalized concentrations for the concentrated trial should have been the same as the diluted trials, but they were consistently below the diluted trials. This was kind of disturbing, and has prompted me to run more concentrated trials. I also updated my results report, and fixed and updated all of the graphs that I have been creating. I also consolidated and organized all of my data and graphs.
Yesterday, I started by running the last trial of the second series. I also prepared a few more tests and ran them as well. The first was a new test for chlorine, which worked slightly better than the first. Part of the problem was that I didn’t prepare the NaCl standard quite right, but some of the results still come out negative. I also ran some of the Chromatin that the professor and Travis have been working on, and found that it had higher calcium concentrations than they expected. I also designed and ran a highly concentrated series of the first series. This should help identify what is actually going on in the last few samples of each trial. They have been fairly random, but almost always are way above where I would think they should be. This is attributable to the fact that are very low concentrations of all of the elements. This makes sense, because less DNA is precipitating.
