Tuesday, June 20, 2017

End of fifth week already?

Time surely flies by when you're working full on. This week, I started working on another batch of ITC experiments after we figured out a number of flaws in our experimental protocols. Therefore, we decided to redo some of our previous experiments using different experimental parameters and protocols.
We decided that we would keep the relative concentrations of DNA and Cobalt Hexammine unaltered in the different ITC runs. The concentrations of DNA and Cobalt Hexammine used in the experiments were 2.5mM DNA+3mM Cobalt Hexammine, 5mM DNA+6mM Cobalt Hexammine, 10mM DNA+12mM Cobalt Hexammine. In addition, we decided to alter the experimental procedure a tad bit. We decided to form isotonic DNA+NaCl solutions using the dialysis method so that the concentrations of NaCl in all the samples are equal. The dialysis buffer was used for the serial dilution of the 120mM stock Cobalt Hexammine (instead of water as in the previous experiments).

After I prepared the batch of DNA samples (of different concentrations) using the regular method, I filled up dialysis tubes with 10ml of each sample. I left the tubes in a dialysis buffer of 10mM NaCl solution overnight. I used the same dialysis buffer for the serial dilution of the 120mM stock of Cobalt Hexammine in order to synthesize 10ml of 12mM,6mM and 3mM solutions respectively.

After the synthesis of the DNA samples and the Cobalt Hexammine solutions, I ran heat of dilution tests on the Cobalt Hexammine samples. Here are the results:

After running the heat of dilution experiments, I proceeded to start ITC runs with my DNA samples. I started off by using my 10mM DNA sample. Here's the overlay for the raw heat data and the integrated heat data:

That's all the highlights from week 5 folks. Stay tuned for more ITC results next week!

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