Monday, July 18, 2016

July 18th - Gels, Gels, and More Gels

For the past several weeks, I have been working on "digesting" my sample, and running sample os varying degrees of digestion with gel electrophoresis. The first step of the process is to do a "trial digest," in which different concentrations of micrococcal nuclease are added to the nucleosomes. Micrococcal nuclease effectively "eats" the DNA, slowing down when it approaches the histone core. A higher concentration of micrococcal nuclease will "eat" more DNA, so the optimal amount that will digest only the linker DNA is sought.

Next, proteinase K is added to the nucleosomes, which digests the histone proteins. There is now free DNA in the sample, and its length is determined by the amount digested by the micrococcal nuclease. Gel electrophoresis can be done on the variously digested samples to qualitatively see how long the DNA is for each micrococcal nuclease concentration (proteinase K concentration stays the same). The goal of this "trial digest" procedure is to determine what concentration of micrococcal nuclease digests just the linker DNA, so that all we are left with in the sample is the histone core and DNA wrapped around it. It is known that there is approximately 146 bp of DNA around a histone core (without the linker DNA).

It took several attempts to get a successful gel though. Examples of successful and unsuccessful gels are below.

This gel is an example of a gel that was not successful. It is a good gel in regards to the quality of the 10 bp DNA ladders (in the first and last lanes), but the samples are all trapped in the wells, rather than moving down the gel like the DNA ladder did. Such behavior could be explained by improper digestion, perhaps because of the micrococcal nuclease itself, its digestive medium, or other factors.

This gel is an example of a successful gel! Although the DNA ladder is not as clear as the gel above, the samples did not stay in the wells, and instead, moved down the gel depending on their length. It is interesting, though, that a majority of the digested samples have a length of approximately 300 bp (double what it should be!).

Because the 40 units of micrococcal nuclease seemed like the optimal concentration to digest the DNA, the sample was digested with this concentration, and compared in another gel to the prior undigested sample (see image below).

This gel contains the DNA ladder (not very clear), the digested sample, and the undigested sample. It is good that there is a stark difference between the digested and undigested sample...but where exactly is the undigested sample? Is there DNA there, is it over digested, or does the concentration of DNA in the gel sample need to be increased?

No comments:

Post a Comment