03/06/13

Today, my mentor taught me how to denature a given sample. Of the steps that I had to do last week and the week before that, this process proved to be the easiest. First, I was told to add a certain measurement of a DNA sample and a certain amount of the formamide mixture. Formamide is a denaturing agent. In addition, my mentor instructed me to add a parcel of fluorescent dye—important for locating the DNA when run through by the electrophoresis capillary machine. (In my case, I added 2 microliters of DNA and dye and 16 microliters of formamide. After creating this mixture in one vial and wrapping it securely with film, I inserted it into water that was heated to a temperature of 95 degrees Celsius for approximately five minutes. While that was taking place, I went downstairs and got a beaker of ice, which would be needed to cool the heated sample. This last process would take 15 minutes in total.

Looking at the big picture, this fits in with the whole concept of capillary electrophoresis as only then will scientists be able to obtain the best accuracy and precision in sizing the results.

03/27/13

Last Wednesday, my mentor tasked me with the usual routine with preparing samples for her to test in the capillary machine. Rather than starting with a new chemical substance however, she had me use the buffer I made the week before as one of the components that make up the sample. After doing the calculations—which are each week getting progressively easier for me to accomplish,— I measured out the required amount of each substance and combined it into one test tube. Knowing that 1000 microliters is equal to 1 milliliter, I made the needed conversions and adjusted the pipettes accordingly. After about one hour, I had my final product: 3 vials and in it, 0.1M, 0.05, and 0.14M of Na₂P₂O₇ respectively.

While the process mentioned thus far was repetitive of that of what I did last week, what came after proved to be somewhat different. Yolanda (my mentor) guided me in adjusting the pH of the samples that I made to that of 7. Although we had briefly talked about this process a week prior—during which I had thought it was simply a trial-and-error procedure—I found it to be far more complicated. Due to the fact that one can’t take out the added liquid (which is used to either make the sample more acidic or basic) once inserted, I had to be very precise with my guesstimations so that I don’t ruin the sample that I had spent creating for the past hour. Because my mentor is more of a “what do you think you are supposed to do” type of person more so than a “here is how you do it” type, I was even more nervous as I did not want to disappoint with a process which was, to her, like counting the alphabet. First I added a medium portion of the other acidic and basic fluids that she prepared before, and based on the change in pH I either doubled or lessened the next amount. After the first two samples however, I quickly attained the feel of the steps and by the third vial, I completed it without her help.

Although the steps I carried out today was similar to that of last weeks, it was not the exact same process. When last week I prepared two buffers, this week, using the very buffers, I created 3 samples, each a mixture of different concentrations of various ingredients. As of now, I have learned how to prepare buffers, measure the pH level of buffers, prepare samples, and achieve a particular pH value for that particular sample. This coming Wednesday, I am curious as to what I will be doing and hopefully sometime soon, my mentor will use the products I created and run them through the capillary machine!