Friday, May 26, 2017

Week 2- More ITC stuff

For those of you wondering, here's what the TA Nano-ITC instrument really looks like!
I had a disastrous start to week 2. On Monday, I was in bed for most of the day with a flu. However, things started to pick up on Tuesday. I started off by running the ITC machine on the DNA sample that I made last week. My job was to make the 3mM DNA-NaCl solution react with the 6mM Cobalt Hexammine solution(also made last week) and record the enthalpy changes that signify the two binding phases of DNA, i.e. the binding of DNA with Cobalt Hexammine and the subsequent condensation of the DNA molecule.

I started my experiment by loading the Nano ITC device with the reactants. I loaded the sample cell of the ITC machine with 300 µl of the DNA solution. Next, I loaded the buret syringe with 50 µl Cobalt Hexammine. The process of loading the buret syringe was extremely difficult and stressful the first time. I had to make sure that there were no air bubbles present in the Cobalt Hexammine solution contained in the syringe column. Not only that, I had to poke in a air bubble at the top of the liquid column using the plunger. I remember sweating profusely while desperately trying to pipette air bubbles out of the column! That was a time consuming process.

The next part was pretty straightforward.

 I just used the ITCRun software installed in the computer connected to the ITC device to operate the machine. I basically set all the parameters required for the reaction to proceed and the software did the rest. After equilibrating the sample solution for about 30 minutes, the ITC started emptying the Cobalt Hexammine,bit by bit, into the sample cell after regular intervals of 175 s. At the same time, the software plotted and recorded enthalpy peaks after every 175 s. 

Sadly, my first trial was a massive fail with the first few plots going horribly wrong. I'm assuming it was due to the presence of a massive air bubble at the tip of the syringe. 
Results from run 1
I re-ran the experiment with the same settings for a second time. Didn't work this time either!

When you fail, you have to try harder. That's what science is all about.
So, I reset the experiment for a third time and started the experiment with fingers crossed. And it worked this time!
Result from run 3
I re-modeled the raw data using the NanoAnalyze software to display the thermodynamic parameters involved with the experiment. This was necessary to prove the presence of two distinct binding phases of DNA. 
Remodeled data from run 3
In order to ensure consistency in the data I ran the experiment once more. The results from this run looked similar to the results from the third. Hence, this run was successful too!

Our next objective was to create a broader spectrum of peaks in between the two binding phases of the DNA. In other words, I had to figure out a way to shift the sigmoid curve towards the right and magnify it. I used the simulation mode in the NanoAnalyze software to devise parameters that would enable us to meet this requirement. I won't go into details about how this simulation mode works. But here is a model that the simulation mode prepared for us:

 It recommended that I use a 5mM DNA solution for my next ITC run. I made a 5mM DNA-NaCl solution by dissolving 16.7 mg of calf thymus DNA in 10 ml NaCl. 

I ran this new DNA solution in the ITC with the 6mM default Cobalt Hexammine solution. Here are the results so far:
Results from 5mM DNA w 6mM Cobalt Hexamine run 1
The raw heat data displays a wider range of peaks between the binding phases. Hence it is safe to say that the experiment was successful. To confirm this, I remodeled the raw data using NanoAnalyze:
A perfect match with the experimental model from the simulation mode! 
This week has been an eventful one. Starting off with a few bad trials, I was eventually able to make things work and obtain much better results. A great end to week 2!

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