02/20/13

Today, as soon as I arrived, I was put to work as to making the two buffers. Applying the knowledge that I learned last week—about finding the correct calculations pertaining to the measurement of the particular substance—, I was asked to find the target amount. When last week, I dealt with a liquid (though only hypothetical because I didn’t actually get to create the mixture), this week, I prepared a buffer using solids: Na₄O₇P₂ (446.06 Mw) and H₂Na₂O₇P₂ (221.94 Mw). Wanting a total volume of 20mL in each of the two buffers that I was going to prepare, I found the weight of the former and latter solutions needed in grams. To do this I used the formula: weight of solute (g)= formula weight of solute (g/mol) x molarity (mol/L) x final volume (L). After finding the measurements 1.7624g and 0.8878g respectively, all was left for me to do was put combine the various components into their vials.

After having prepared this, Yolanda told me that we were looking to attain a mixture of the two buffers with a pH of 7. Unfortunately, we were not able continue with the next steps because the Na₄O₇P₂ did not dissolve with the H₂0 as expected. This said, my mentor did walk me through the steps that we would have performed had the substance dissolved. By using the pH measuring apparatus, we would—similar to that of trial-&-error—pour in a new vial a portion of the H₂Na₂O₇P₂, which had a pH of 10, and Na₄O₇P₂, which had a pH of 6. Using the pH machine, we would add a little bit of each until the number 7 pops on the screen, thus indicating us that our target goal has been achieved.

In a broader scope, this pertains to the entire goal of capillary electrophoresis (electrophoresis separates macromolecules by size, charge, and/or properties) as a buffer is needed for the conduction of charge. This charge is transmitted by the ions provided by the buffer. In addition, “the buffer, by providing a reservoir of weak acid and base, also keeps the pH within a narrow range. This is important because the structure and charge of a protein or nucleic acid will change if subjected to significant pH changes, thus preventing proper separation.”

Next week, my mentor and I are hoping that the substance will have dissolved and then go from there to continue what we left unfinished today.

The Purpose of Buffer in Electrophoresis. Retrieved from http://www.ehow.com/about_6613320_purpose-buffer-electrophoresis.html.

02/13/13

       On February 13, rather than my normal prepare-sample-weekly routine, my mentor gave me a one-on-one lesson on chemistry. Because next week, she wanted me to not only just measure the pH level of the buffers, but actually prepare them all by myself, I had to be sure that I knew how much of one substance I had to mix with another and the likes.

     To begin with, my mentor first gave the the molecular weight, density, and w/w of the compound HDTMP. In addition to this, she gave me the concentration measurements of the chemical tris and DETA: 200mM and 20 mM respectively. Informing me that we wanted our end product to have a volume of 6mL with Tris being 15mM, DETA: 0.2mM, and HDTMP each at 0.06 & 0.08, she asked me how much of each substance I would have to allot and mix together to produce the two final 6mL product. Being bombarded with all this chemistry all at once, I immediately felt lost and like that of my first internship day, I felt like my mentor was once again, speaking in another language. It had been more than a year that I had last learned or thought this heavily about chemistry and this painful (and embarrassing) experience had me realize that science is not only just biology, or just only chemistry, but a combination of all the subfields merged together to create a whole. 

      However, after a while, I eventually caught on and the things learned last year in chem class started to come back to me little by little. Using the C1V1 = C2V2 formula, (aka V1= C2V2/ C1), I was able to plug-and-chug the numbers and figure out (to my great relief) the proportions for the amount of volume needed for Tris and DETA chemicals. (This is a gross exaggeration of the mathematical process that I had to do. It's way harder than it sounds.) In addition to this, I had to pay heed to the units and change it from cm^3 to mols per liter to mL. 

    Although I had learned of the basics last year, the problem that I was tasked with was chemistry to another level. Math and chemistry not being my forte, I struggled majorly to keep up with my mentor's explanations, but by the end of our time together, I fully understood and new what to do the next time around when asked a similar problem as the one that we solved together. Despite this day being of a chemistry class, I was glad to have emphasized my lacking foundations as chemistry was/is an important part of any sub-field of science in the past, present, and in the ongoing future.

        This coming Wednesday, I will be *gulp* expected to first calculate the correct measurements and then be expected to, all by myself, create the needed buffers.

02/06/13

Today, I prepared DNA samples for my mentor. When last week, I prepared the buffers to be inserted in the capillary machine, this week, I held the responsibility of producing the main ingredient: the DNA samples.

Using different types of 76mers –single-strand DNAs that contain 76 bases–, I first created a mixture and put it in a sample tube. This tube contained a combination of 5 microliters of each 76mer. Next, I allotted to 10 separate tubes, 2 microliters of this very mixture. Because it would be the same product, if this mixture was simply put in the capillary electrophoresis machine, one would get a graph with about the same peak levels for each DNA. Given the fact that our end goal is to find out the specific location of the particular 76mer within the mixture, my next step was to add 0.4 microliters of each unique 76mer into the 10 separate containers. This 0.4 microliters is called the “spiking sample” as it will increase 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. After all this, I added Formamide—a denaturing compound—used to separate potential dimers/hair-pins created between the single-stranded 76mers. This step is necessary as the DNA sample has to be kept single-stranded in order for the bases (adenine, guanine, thymine, cytosine) to base pair with the DNA. These base pairs will then help differentiate the speed at which the DNA sample hits the cathode end of the capillary electrophoresis machine. Later on, my mentor will use these very samples to figure out the sequences of each of the 76mers based on the graph that will be produced from the capillary electrophoresis machine. 

All in all, I am looking forward to seeing how the samples I prepared for the first time actually turn out next week! 

01/30/13

        Today, not only was I finally able to attend my internship, but also I was able to partake in the lab process! I helped my mentor Yolanda prepare her test DNA tubes that we later inserted in the electrophoresis capillary machine. 

       In addition, today I was introduced to the pH meter: an electronic device used for measuring the pH of a liquid. This meter consisted of a glass electrode (a special measuring probe) connected to an electronic meter that measures and displays the pH level. The pH probe measures the concentration of Hydrogen ions in a given solution in moles per liter. (In other words, it measures how acidic or basic a solution is). Prior to using, Yolana informed me that I must always calibrate the meter and that after each measurement the probe must be rinsed with distilled water to remove any traces of the other solution measured. Calibration should be performed with at least two standard buffer solutions that include the range of pH values to be measured. The glass probe when in use, produces a small voltage that is then measured and shown as pH units by the instrument. (approximately 0.06 volts per pH unit). With this tool, I was given the task of finding and then labeling the pH levels of the 8 buffers that Yolanda had made just the day before.

       This Wednesday, I am going to further assist my mentor with her research. Because last week there was an unfortunate problem with the machinery, we weren't able to get the predicted results. Thus, I am hoping that this time, everything will go as planned.

01/23/13

I could not go this Wednesday because of MLK day.
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 What is Electrophoresis in general?

Electrophoresis, according the Wikipedia is “the motion of dispersed particles relative to a fluid under the influence of a spatially uniform electric field.” Expressed in layman’s terms, this can be translated to the definition: the movement of electrically charged particles in a fluid under the influence of an electric field. This phenomenon was observed first by Ferdinand Frederic Reuss in 1807. He noticed that the application of a constant electric field resulted in clay particles mixed in water to migrate.

The electrophoresis of positively charged particles is called cataphoresis. In a similar sense, the electrophoresis of negatively charged particles is called anaphoresis. Electrophoresis is used anytime a separation of molecules is needed. For example, DNA electrophoresis is used to study the genetic makeup of plants, animals, and humans. This is an analytical method used frequently in molecular biology and medicine as it is applied for the separation and characterization of nucleic acids, proteins, viruses, small organelles, and the likes.

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

01/16/13

My ride to RPI was cancelled because of the snow.

__________
Capillary Zone Electrophoresis

Due to the fact that I could not go to my internship again, this week, I did my research on CZE. 

(While researching this topic, I realized over and over again how hard and challenging science is. Almost all of the scientific terms I had rarely heard of, and understanding what I was reading required a lot of research.)

The simplest form of CE is capillary zone electrophoresis (CZE), also known as free solution capillary (FSCE). This form is based on the differences in the charge-to-mass ratio. For this method of electrophoresis, all that is needed is a “well-chosen buffer”. Separation is able to take place because of the relatively simple interaction of the analytes with the pH of the buffer.

The CZE samples being analyzed move in the EOF (electroosmotic flow: “the movement of ions through a solute under the control of an applied potential") but then separate into different bands as a result in the differences in their electrophoretic mobilities, µ. (Electric mobility is the speed at which macromolecules pass through a matrix “in the presence of the electric field”)

The differences in µ make each analyte’s overall velocity slightly different. This difference in velocity is also known as separation.  

The steps required for CZE are very straightforward. First, one washes the capillary with buffer. Then, the sample—already dissolved in the same buffer—is injected and the EOF is established.  

Capillary zone electrophoresis is extremely useful for the separation of peptides and proteins since “complete resolution can often be obtained for analyses differing by

only one amino acid substituent.” While researching, I found it particularly interesting that this is very important in tryptic mapping where mutations and post-translational modifications can be found.

Beckman Coulter. Introduction to Capillary Electrophoresis. Retrieved from

https://www.beckmancoulter.com/wsrportal/bibliography?docname=360643-CEPrimer1.pdf.

Chasteen, T. F. Modes of Electrophoresis. Retrieved from http://www.shsu.edu/-chm_tgc/primers/pdf/CES.pdf.