04/24/13

At last! I got the results that I have been waiting for!

Today, I was able to see the final result from the electrophoresis machine. In the graph below, the peaks represent the corresponding 76-mers mentioned in my prior blog. (76-mers are single stranded DNAs that contain 76 basers). A while back, I prepared a similar sample for Yolanda with our end goal to find out the specific location of the particular 76mer within the prepared solution. Because this sample consists of a "spiking sample" (this increases the concentration of that particular 76mer in that particular tube so that when inserted into the machine and read, the graph will indicate the precise location with a peak that is higher than the other 9 polymers present in the mixture), my mentor and I were able to see that the spiked sample has 3 times the concentration of the other 76-mers--the highest peak being this very spiked peak.

In retrospection, I can't believe I have finally completed the whole process of capillary electrophoresis. Starting from learning about the chemistry of it all to actually preparing the buffers and samples, and then finally getting to use the capillary machine, I have learned so much from this experience. Next week, which I believe will be my final week, I am hoping we will be able to see more graphs like the one below and discover exciting results.

04/17/13

         Finally!! Today during my internship I was introduced/ got to use the capillary electrophoresis machine! Just because it was so long since I last wrote about the machine, I will give you a recap: In electrophoresis, proteins are separated and then characterized according to various characteristics (ie: size and charge) when moved across an applied electric field. Once completely separated, scientists are able to find abnormalities according to the types and densities of proteins in the sample. Consequently, they are then able to diagnose disease conditions. Unlike gel electrophoresis (in which the medium used for separation is a gel), capillary electrophoresis uses a liquid buffer <which now I am extremely good at preparing :) >. This gives the protein the ability to move more naturally and freely as compared to migrating through a gel. In short, capillary electrophoresis is a technique that separates ions with the use of an applied voltage.

 Today, I learned everything from how to start the machine, how and where to insert the buffers and DNA sample, how to run a test trial, and so on. Each side of the high volage supply is connected to an electrode which helps induce an electric field to initiate the movement of the sample from the anode to the cathode via the capillary tube. First, the capillary tube must be coated with the prepared buffer solution. "Electroosmotic flow is observed when an electric field is applied to a solution in a capillary that has fixed charges on its interior wall. When a buffer solution is placed inside the capillary, the charge is accumulated on the inner surface." Thus, before the sample DNA is inserted for reading, the capillary must be coated with the desired buffer.

     After the run through of the machine, using the buffer I prepared and the sample that my mentor pre-made, I got to use the machine. Putting everything together was not hard [this is one of the many advantages of capillary electrophoresis] as all I had to do was first rinse the tubes with water [cleaning it of remnants of the buffer solution from other runs] and then placing the two viles of buffer and sample in their respective places. I also made sure the temperature of the machine was what it needed to be as extreme temperatures could cause many malfunctions, ie: causing proteins to denature. Then, my mentor showed me how to take a reading of the sample via the hooked up computer. Because the explanation process concerning the workings of the machine and preparing another set of buffers took a fair amount of time, I did not get to see the produced graph (as it takes 30 minutes or more to produce 1 graph).

However, this coming Wednesday, rather than preparing the mixtures, I believe (and hope) I will get to spend more time using the capillary electrophoresis machine and learn how to interpret the resulting graph!

http://www.chem.sc.edu/faculty/morgan/lab.html

04/10/13

Today at my internship, I prepared yet another set of buffers. (I have made so many buffers now that it has become a procedure in which I can carry through while sleeping!) When last week, however, my buffers were created from solids (the various powdery substances), today, the six buffers I was told to prepare was created from liquids. The three were: CH₃COONa, NH₄HCO₃ and (NH)₂SO₄. I was told to measure out 0.4 mL and 0.2 mL of CH₃COONa and NH₄HCO₃ while measuring out 1.68 mL and 2.4 mL of (NH)₂SO₄. After this process, I diluted the solutes with distilled water to get a final volume of 6 mL for each of the six tubes I prepared. [all had a molarity of 0.5M]

            Once these steps were done, I quickly calibrated the pH meter using three beakers containing solutes with pH 7, 9 and 10 respectively. After this preliminary step was completed, I measured the pH levels of the six solvents I had concocted and labeled them accordingly. (In the end, the pH levels of all 6 tubes came about roughly to the estimated 8)

Today, like the week before, I spent the entire time consumed with preparing these buffers. In retrospection, I can definitely say that my science capability and knowledge—more specifically that my laboratory skills—has improved tremendously. My current capability to go in and prepare a buffer or a test solvent based on measurements (numbers) alone never fails to amaze me. Next week, I am hoping to be doing something other than preparing buffers and actually moving towards working with the capillary electrophoresis machine.

04/03/13

                During this recent Wednesday, I went to my internship and was tasked with making a new group of buffers that my mentor would use in her later capillary electrophoresis test-runs. This time, she had me mix up five different buffers using 5 different powdery (solid) components: 6-phosphoneohexanoic, 1.10 decyldiphosphonic, n-phosphonomethyl glycine, aa, and COO. Using a balance beam, I measured out precisely the indicated grams and afterwards, mixed each of the 5 separate beakers with the liquid mixture tris. After all this, I then put the beakers one by one on top of a hot-plate magnetic stirrer device in order to mix fully dissolve the powdery components into a liquid solute. This was my first time both seeing and using this particular device and thus I was greatly amazed. Although there is not much to the  instrument--the left knob controls the stirring rate and the right knob controls heating--I found it to be an extremely useful device that could be used in a variety of situations in the laboratory. First, I added a small magnetic stir bar in the mixing solution. Since glass doesn't affect a magnetic field and most chemical reactions take place in glassware (ie: beaker), magnetic stir bars work well in almost all circumstances. However, the downfall is that the limited size of the bars restricts the effect of the instrument to relatively small (ie: under 4 liters) experiments. For larger volumes, another sort of stirring device is needed. This said, I was asked to make buffers with a volume of 3 liters so the stirring bar proved extremely helpful to me. After I had done all this, my next step was to set all of the buffers to the pH value of 7 (given to me prior from Yolanda, my mentor) using the methods used during my last internship before spring break.

Today unlike my last time here, I found myself working hard until the very end, trying to accomplish everything that I had been assigned. Although that meant standing up for the entire 3 hours, I fully enjoyed yet another day in the lab. I was able to experience first-hand, like any typical scientist, truly how fast time passes by when fully engrossed with one's laboratory procedures. I have yet to actually see or put my prepared solutes/substances in the capillary electrophoresis machine, but I am hoping that within the next couple of weeks, the opportunity will come up.

Above is a short clip of the magnetic stirrer that I very much enjoyed using.

03/13/13

I did not go this Wednesday as my mentor had a conference that she needed to attend. However, I went ahead and decided to learn more about the substance Tris, that I always use when at the laboratory making solutes for my mentor.

Tris (aka THAM) is the abbreviation for the organic compound: tris(hydroxymethyl)aminomethane. It's formula is (HOCH2)3CNH2. In biochemistry--ie: the type of science my internship involves--this compound is widely used as a component of buffer solutions, especially for solutions of nucleic acids. It has an acid dissociation content (pKa) of 8.07 at 25 degrees Celsius, which implies that its effective buffer pH range is between 7.0 and 9.0.

An acid dissociation constant, Ka, (aka acidity constant, or acid-ionization constant) is a quantitative measure of the strength of an acid in a solute. It is the equilibrium for the chemical reaction: dissociation in the context of acid-base reactions. [dissociation is a process in which ionic compounds separate into smaller particles or ions] (ie: KCL <--> K+ + Cl-)

Tris inhibits a variety of enzymes and thus should be used very carefully when studying proteins. However, this compound is one of the most widely used buffers in molecular biology and cell culture as a result of its low stability, toxicity, & buffering capacity. (In addition, Tris can be obtained at a low cost, in relation to the other components used in laboratories)

Tris. Retrieved from http://en.wikipedia.org/wiki/Tris.

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!