2017 EMCC Student Conference

Similar to last week, this week consisted of working on my presentation and continuing to revise my poster. Not only that but I actually presented my poster (attached below)! I reminded myself that whatever happened, would happen. Either I would place in 1st, 2nd, or 3rd or I wouldn’t place in any at all. Quite frankly, I was okay with that because I was satisfied with what I had brought to the table. Plus, with the assurance of my mentors, Josh and Matt, and Arane, a friend of mine, I slowly started to gain confidence. In the end, I did not receive any award but Arane placed in 1st! :) I’m proud of myself for making it into the conference and coming this far. I know there is more waiting for me!

DNA Extraction from Enterococcus faecalis Using Four Different DNA Extraction Methods.

Ready to begin presenting my research project at the 2017 EMCC Student Conference.

Presenting my research project in front of the 3 judges at the 2017 EMCC Student Conference.

EMCC Student Conference Poster

I was fortunate enough to have been selected to present my research project at the 2017 EMCC Student Conference. Because of this, most of my week consisted of adding, editing and deleting items from my poster. Creating a poster may not seem like fun for others but in this case, I enjoyed making it. In high school, I took a graphic design class (which I loved) and even though this poster did not require much, it definitely reminded me of that class. It reminded me of the way colors work, the spacing between words and how small or big images should be. This was different than the usual “researching and following protocols” day. Now, that’s not to say I don’t like those days but I definitely liked the change in schedule. Below is a draft of the poster (I will upload the final once it has been revised/ approved). Nonetheless, presentations begin on April 26th and I am trying not to freak out!

2017 EMCC Student Conference Poster Draft

Electrophoresis

This week, to determine the quantity of DNA yielded from each extraction method, (QuickExtract DNA extraction kit, boiling, microwave and enzymatic digestion followed by heating) agarose electrophoresis was performed. Through this, I became very confused with the results. Before I get into this, in the first slot, I had inserted a PCR MW Ruler. After that, the following slots from 2-13 were filled with 1 microliter of dye mixed with 5 microliters of DNA from QE1 (QuickExtract protocol), QE2, QE3, M2 (microwave protocol), M3-1, M3-2, M4, B3 (boiling protocol), EDL2 (enzymatic digestion followed by heating using Ready-Lyse Lysozyme), EDL3, EDL4, EDL5 and EDP1 (enzymatic digestion followed by heating using proteinase K) respectively. Using the NanoDrop spectrophotometer (Figures 1 and 2), it is shown that the enzymatic followed by digestion samples had relatively a higher amount of DNA (looking under the column “nucleic acid”). With that said, I was expecting that slots 10 and beyond (which had the enzymatic digestion followed by heating samples) would have bright strands of DNA. The brighter the strands, the more DNA would be present. However, to my surprise, that was not the case. Instead, the microwave method, which had less DNA than mentioned protocol, had brighter strands. As a matter of fact, slots 9-14 appear to have no strands at all (Figure 3). Next week, PCR will be performed on these samples to determine if I have any DNA. In the meantime, I am hoping that the PCR will not cause any further confusion.

Figure 1. NanoDrop Spectrophotometer nucleic acid report of all samples from different DNA extraction methods.

Figure 2. NanoDrop Spectrophotometer nucleic acid report of all samples from different DNA extraction methods.

Figur,e 3. Electrophoresis results after sampling PCR MW Ruler, QE1, QE2, QE3, M2, M3-1, M3-2, M4, B3, EDL2, EDL3, EDL4, EDL5 and EDP1 in each slot respectively.

Enzymatic Digestion Followed By Heating Protocol Modifications

After looking at the Nucleic Acid Report I collected from the NanoDrop Spectrophotometer last week, I noticed that the best DNA extraction method (compared to the others) so far is the enzymatic digestion followed by heating using Ready-Lyse Lysozyme and proteinase K (Figure 1). When referring to the “best DNA extraction method”, it is the protocol that has a purity close to 1.8 for DNA and close to 2.0 for RNA under the 260/280 column (“260/280 and 260/230 Ratios”, n.d.). Because the values for the enzymatic digestion followed by heating using Ready-Lyse Lysozyme and proteinase K are somewhat close to these values (closer than the other protocols, excluding the commercial kit), during this week, I worked specifically with these two protocols. However, the past week, in tubes EDL1, EDL2 and EDL3, I added 1 microliter of Ready-Lyse Lysozyme and in tubes EDP1, EDP2 and EDP3, I added 2 microliters of proteinase K. This week, in tubes EDL 4 and EDL 5, I added 2 microliters and 3 microliters of Ready-Lyse Lysozyme respectively. Using proteinase K, in tubes EDP 4 and EDP 5, I added 3 microliters and 4 microliters of proteinase K respectively. The results of the purity (260/280 column) of DNA in each tube are shown below in Figure 2. With these results, the increase in Ready-Lyse Lysozyme seems to decrease the purity of DNA and RNA and decrease the number of nucleic acid. However, an increase in proteinase K seems to have very little effect on the amount of nucleic acid and purity of DNA and RNA, increasing slightly in both areas.

Works Cited:

260/280 and 260/230 Ratios [PDF]. (n.d.). Wilmington: Thermo Scientific.

Figure 1. Eppendorf tubes EDL1, EDL2, EDL3, EDP1, EDP2 & EDP3 used in the enzymatic digestion followed by heating protocol using Ready-Lyse Lysozyme and proteinase K.

Figure 2. Eppendorf tubes EDL4, EDL5, EDP4 & EDP5 used in the enzymatic digestion followed by heating protocol using proteinase K.