Starting PCR

After waiting a week for the primers to arrive, I was finally able to start PCR. However, before I began, I had to prepare my primers. By doing so, I had to centrifuge them, add the designated amount of Tris Hydrochloride to each primer (Figure 1) and shake like crazy! This process took about 4 hours because I had to make sure my primers were completely mixed with the Tris Hydrochloride (thus, ready to use). After I prepared my primers, I set up my samples for PCR. Each PCR tube required 10 microliters of 2X Master Mix for PCR, 2 microliters of the forward primer, 2 microliters of the reverse primer, 4 microliters of sterile water and 2 microliters of DNA (from my bacteria samples). For this PCR run, I used the PCAT-4f-2015 and PCAT-4r-2015 primers (they are a set). After I had all of my samples, I set them into the PCR machine, ran the “PAUL” PCR protocol and collected them when they were finished (Figure 2). Next week, I will be using the next set of primers: 27F and 1492R.

Figure 1. From left to right, PCAT-4r-2015, PCAT-4f-2015, 27F, 1492R and Tris Hydrochloride. The first 4 are the primers I will be working with and the last item is the solvent the primers needed to be dissolved in.

Figure 2. Collected samples after PCR was complete. The DNA samples used were from Group A (except for #12, which was from Group C). 

Unknown Creosote Bush Bacteria

This week, I performed a third and final DNA extraction (Group C). These values varied from the last two (Group A and Group B) because some values were high and others were low. Considering that I have enough DNA from Group A to perform PCR, I decided to call it quits after I received the data from Group C. Attached below is the nucleic data from Group A, B and C. Aside from that, because I cannot perform PCR without master primers (which will arrive next week and is my next step), I retrieved a leaf from a creosote bush and carried out tests to try to determine what kind of bacteria was present. I liked running these tests because not only are they simple but they reminded me of my first semester at S-STEM. Sometime this week, I will hopefully be able to determine what kind of bacteria I’m dealing with!

Figure 1. The first DNA extraction group (Group A) from unknown bacteria, which had relatively high nucleic acid (DNA) compared to the others.

Figure 2. The second DNA extraction group (Group B) from unknown bacteria, which had relatively high nucleic acid (DNA) compared to the others.

Figure 3. The third DNA extraction group (Group C) from unknown bacteria, which had relatively high and low nucleic acid (DNA) compared to the others.

Figure 4. The graph of the third DNA extraction results (group C) displaying how much was absorbed during a certain wavelength. The colorful numbers to the left side correspond to the bacteria it is. "EC" stands for E.Coli, which is being used as the positive control. Note that the absorbance goes up to about 32.

Figure 5. Unknown bacteria from a creosote bush under the microscope after gram staining. This bacteria is gram + (because it is purple) and cocci shaped (the cell shape is circular).

Bump on the Road

For some reason, I didn’t feel like myself this week because I would do little mistakes here and there (which I do my best to avoid). For example, when working on the QuickExtract DNA Extraction protocol, I would make a few mistakes that made a huge difference. For instance, the first step after centrifuging the bacteria is to remove the supernatant and then add sterile water. Instead, I did not remove the supernatant and added the sterile water, causing it to mix. This resulted in a low amount of DNA (Figure 1). I committed another mistake when I attempted to fix my first one. This time, I lowered the amount of bacteria I used and immediately put away my finished DNA in the refrigerator (when it should be left at room temperature for awhile). This error gave me an even less amount of DNA (Figure 2). Of course, I was bummed out (and still am) but I will be returning to take my time and pay close attention to what I’m doing. As the saying goes, you learn from your mistakes!

Figure 1. The graph of the first DNA extraction results (group A) displaying how much was absorbed during a certain wavelength. The colorful numbers to the left side correspond to the bacteria it is. "EC" stands for E.Coli, which is being used as the positive control. Note that the absorbance goes up to 40.

Figure 2. The graph of the second DNA extraction results (group B) displaying how much was absorbed during a certain wavelength. The colorful numbers to the left side correspond to the bacteria it is. Note that the absorbance goes up to 7.

QuickExtract Protocol

This week, I focused on transferring grown bacteria from the TSA plates into a broth and then performing a DNA extraction method (shown below). The protocol used was the QuickExtract Bacterial DNA Extraction Kit, which was the control in my project last semester (it gave me the most amount of DNA). Like it’s name, this method is quick and quiet simple to set-up. However, one aspect that I do not like is that when all the steps have been completed, the final resulting “liquid” (DNA) is cloudy. In order for it to be used in the NanoDrop or for PCR, the liquid has to become clear, which, unluckily for me, can take up to hours or days. From the 14 bacteria I’m working with, only test tubes #4, #13 and #14 became clear in about 4 hours. Unfortunately, I was unable to work with bacteria #2 (it still has not grown on the TSA plate) and #12 (it was contaminated). Hopefully by next week, I will begin working with #2 and #12 to then move closer to finding the DNA of all the bacteria!

Figure 1. 14 bacteria in TSA broth at 0 hours

Figure 2. 14 bacteria in TSA broth after 24 hours

Figure 3. 12 1.7 mL test tubes filled with bacteria ready to centrifuge at 5000 RPM for 5 minutes.