Wednesday, May 24, 2017

Week #2 MTW

So like I last mentioned, I did start off Monday by characterizing the DNA in Professor Thompson's UV-Vis machine. The peak wavelength was at 527nm, this is a good sign. A solution with this peak wavelength indicates that the majority of the gold nano-particles are the size and shape that we want them to be. I then used a quartz cuvette in the UV-Vis machine to examine the DNA. The peak was then at 258nm which is a good place for the DNA's peak to be. We also recorded the wavelengths at 260nm and 320nm. This was done because subtracting the 320 wavelength from the 260 wavelength can allow us to calculate the concentration of the DNA in the solution.

Next I began preparing for equilibrium dialysis. First I prepared some TEM buffer, which is like TE buffer but slightly different. It contains NaCl, Tris, EDTA, and water. This would be what is used to re-hydrate the solutions during dialysis. Then I did more calculations to find the proper ratio to mix the DNA and gold nano-particles together. Unfortunately (?) something must have gone awry, for the DNA and gold nano-particle solutions (which all SHOULD have contained exactly the same thing) looked very different.

It's very clear that these solutions are not all the same. We decided to re-run these samples in the UV-Vis to try to identify where things had gone wrong. The UV-Vis showed us that the samples were all relatively the same, so we continued to equilibrium dialysis. For equilibrium dialysis we use big centrifuges located in Professor Thompson's lab. The procedure calls for 40 minutes at 3000rpm. Unfortunately, I ran the first 40 minutes at 3000rpx (?) which is roughly 5000rpm. This is much more forcible then what should be used, and likely caused the DNA to crash out. After the first spin it looked like this:

To me, this looked pretty normal. The pellet is not very big, but is definitely noticeable. The supernatant was then siphoned off and the solution was rehydrated with TEM buffer. The second run, I was sure to set it at 3000rpm for 40 minutes. After the second run the solution looked like this:

It is very noticeable how small the pellets are and this concerned me. I then siphoned off the supernatant and re-hydrated the solution with TEM buffer and ran the solution at 3000rpm for 40 minutes in the centrifuge one last time. After this last run, the pellet was almost unnoticeable. Regardless, I siphoned off the supernatant and re-hydrated the solution. Because the pellets were so small on the second and third runs, we figured that something was wrong. To try to find what had gone wrong, we decided to run everything in the UV-Vis machine again. After getting the results back, it appears that the first run at 3000rxm had in fact caused the DNA to crash out.

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