Friday, May 19, 2017

Week 1- Isothermal Titration Calorimetry

The first week of my research with Dr. Andresen started with some detailed study about the Nano Isothermal Titration Calorimetry (ITC) technology. The basic idea of our research project is to figure out the thermodynamics associated with DNA condensation by Cobalt (III) hexamine binding. Fundamentally, we are trying to reproduce the results from another similar experiment that deals with the enthalpy change of DNA condensation.

Day 1: Monday, 15th May, 2017

I started out by watching youtube videos on how the Nano ITC machine works. ITC is a technique that deals with a wide variety of bimolecular interactions. It directly measures hear either released or absorbed during a biomolecular binding event and it is extremely sensitive to very small heat changes. The Nano ITC machine consists of two identical cells, made of gold due to its inertness and high thermal conductivity. These cells are surrounded by an adiabatic jacket. The instrument uses two very sensitive thermocouples, one in each cell to constantly monitor the temperatures of the cells. The temperatures of the cells are kept equal. The heat supplied to the sample cell will be lass than the heat supplied to the reference cell if the reaction is exothermic and vice versa.

Day 2: Wednesday, 17th May, 2017

Wednesday was all about synthesizing the chemicals required for measuring the heat of dilution using the ITC machine. We were to measure the heat evolving from the dilution of cobalt hexamine using 10 mM NaCl solution. I started by making a 0.2 M Cobalt Hexamine stock solution. I then used the 0.2 M stock solution to make 10 ml of 6mM Cobalt Hexamine solution. Subsequently, I made 10 ml of 10 mM NaCl solution from a 1 M stock by dilution with water. I split the 10 ml solution into two tubes, one with 2 ml of NaCl and the other with 8 ml of NaCl (for the DNA solution).

Day 3: Thursday, 18th May, 2017

On Thursday, I used the Nano ITC machine to measure the enthalpy of dilution of cobalt hexamine. I had to be extremely careful while loading the syringe with 50 microliters 6mM Cobalt Hexamine, to make sure that there were no air bubbles inside. Fontaine helped me with loading the reference cell with 300 microliters of water and the sample cell with 300 microliters of  10mM NaCl solution. I had to be extremely cautious so that there were no air bubbles inside the cells. The presence of air bubbles in cells or the syringe can give erroneous results. The injection interval for the cobalt hexamine injection was set to 175 s, 20 injections in total. The ITCRun software was used to operate the instrument. The spin rate was set at 250 rpm and the temperature was held constant at 25 C. The solutions were allowed to auto equilibrate for 1564 s. After that, the syringe automatically started injecting 2.5 microliters of Cobalt hexamine solution after every 175 s intervals. The NanoAnalyze software was used to measure enthalpy peaks after each injection:

As evident from the NanoAnalyze model, the peaks are more or less overtime (except for that last peak which we discarded as an experimental anomaly). The heat evolved after each injection is measured by integrating the area under the curve. The normalized fit for integrated heat vs number of injections was also plotted using NanoAnalyze. Turns out, the normalized fit is not exactly sigmoid as we would expect it to be.

Day 4: Friday, 19th May, 2017

After the satisfactory results from our first test run of the ITC instrument, we decided to start making the DNA-NaCl solution with a concentration of 1 mg/ml. Dylan guided me through the processes of measuring out 8 mg of DNA using the analytical balance, dissolving the DNA in 8 ml of 10 mM NaCl solution and subsequently setting up the solution for DNA shearing. After we completed the process of DNA shearing, we refrigerated the resulting solution. A few hours later, I checked the pH of the solution for consistency. I measured the pH to be somewhere between 6 and 7. The next step would be to measure the enthalpy change of DNA condensation after it reacts with Cobalt Hexamine in the Nano ITC instrument (to be done on Monday). Earlier that day, Dylan also introduced me and Jose to the UV-Vis spectrophotometer and he showed us how to operate the device. Dylan also made demonstrations on how to operate the DLS machine. 

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