Friday, June 17, 2016

Calf Thymus DNA

I started this week by cleaning and concentrating the nanoparticles that I made last week. On Wednesday I finally had my DNA to play with and I started by reconstituting it in TE buffer. The DNA I am using is a very common and cheap type of DNA extracted from a calf's thymus. It can be anywhere from 8-15 kb in length so its too long to make it through the centrifugal filters I have used previously for the PSS. I made 10 mg/mL stocks of the DNA and passed it through a syringe to hopefully shear it some and reduce the viscosity. After I reconstituted the DNA, I titrated into some of the gold nanoparticles at concentration ranging from 0.1 micrograms/mL to 1000 micrograms/mL and measured the UV-vis spectra at each concentration (see graph below).
We noticed that there was positive shift in wavelength with increasing DNA concentration which could be an indication of the DNA wrapping. We also saw some severe aggregation starting with 10 micrograms/mL that could be an indication of the formation of some sort of superstructure arrangement of the nanoparticles on the DNA. It will be possible to confirm if such a structure exists if we are able to visualise the nanoparticles on the TEM. However, the TEM is under maintenance right now so it is hard to now for sure. One superstructure that is possible and has been the subject of a lot of literature is a model histone (see picture below) which would have interesting implications.
I made up solutions of varying concentrations between 1 microgram/mL and 10 microgram/mL and measured their spectra to find a concentration that has DNA binding but no aggregation. The spectra showed the same increase in wavelength with increasing concentration but it also showed that the wavelength actually starts decreasing between 8 micrograms/mL and 10 micrograms/mL. It also shows that 2 micrograms/mL has a noticeable shift with little to no aggregation (See graph below).
Next week. I will attempt to clean off the excess DNA and measure both the bound and unbound DNA in the solutions.

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