Update on preparation of nucleosomes

I am about 4 weeks into research (half way done with my research here on campus for the summer) and so close to having mononucleosomes. Since the last time I posted, we have made progress on preparing nucleosomes. The past couple of weeks have consisted of digestion processes with the DNA and running gels to try to figure out if we had nucleosomes in our samples.

Figure 1: Column Trace of NCP (nucleosome core particle) prep samples

Last week, Dr. Andresen ran a column with the samples we have of DNA. Figure 1 shows the results of the column which helps us determine what each sample has. Each green number indicates a sample number. Thus, we can conclude that samples 1-9 have di and trinucleosomes, where as samples 10-14 have mononucleosomes and the other samples have left over DNA. Therefore, from the column trace we were able to determine that we have mononucleosomes and which samples they are in.  

Figure 2: NCP prep gel run with DNA ladder

Earlier this week I prepared samples to run through 1.2% Agarose gel. As shown in figure 2, we ran 18 samples (4-21) with two DNA ladders on each end. We ran the DNA ladders with the samples because the DNA ladders indicate the length of the nucleosomes. As you can see in figure 2, the red lines indicate how many base pairs there are in the samples, thus distinguishing whether there are  mono/di or tri nucleosomes in the samples. As we had assumed, samples 10-14 most likely have mononucleosomes. We were able to conclude this because one nucleosome has ~147 base pairs and the gel we ran shows us that those samples are closest to having the length of a mononucleosome. Even so, Dr. Andresen is going to discuss our results with Dr. Beuttner, in the chemistry department, in order to confirm what samples we should use to proceed. Even so, I believe that we will continue to purify the samples to try to get as many mononucleosomes as we can. 

Also, now that we are closer to having nucleosomes, I am working on learning how to use the Inductively coupled plasma-optical emission spectrometry machine in the lab. The ICP-OES machine is used to determine the concentration of elements in samples. Thus, I have been preparing samples to run test trials and familiarize myself with the machine as I will be using it to run actual experiments with the nucleosomes.