Electrophoresis & PCR

Honestly, I am trying to stay hopeful because I already want to identify what species of bacteria I am dealing with, even though I am having a bit of a tough time doing so. However, science isn’t always perfect and adjustments have to be made to further in research, even if they’re small. With that said, patience is definitely key in this project. Similar to the past few weeks, I performed PCR and electrophoresis. I ran my post-PCR samples from last week in a gel and the results are shown below in Figure 1. These samples contained 1 microliter of DNA and 5 microliters of water and even though most of the slots showed DNA, there were a few that didn’t. I then decided to switch primers, from the PCAT set to the 27F/1492R set. There’s a possibility that the reason the DNA isn’t showing is because it doesn’t work in the PCAT primer. In figure 2 are post-PCR samples in the 27F/1492R primer set ran under electrophoresis. Luckily, slot 11 had DNA amplification but the others still weren’t successful. Next week, I will continue using the 27F/1492R primer set for PCR and double the amount of master mix, primers and sterile water to have a total volume of 40 microliters, instead of the usual 20. The amount of DNA in each PCR sample will stay at 1 microliter.

Figure 1. Electrophoresis results when adding post-PCR samples into each slot in the PCAT primer set. From left to right, the material in the slots are: MW Ruler, 1, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, and 14. The bands that are shown could possibly be the primers. 

Figure 2. Electrophoresis results when adding post-PCR samples into each slot in the 27F/1492R primer set. From left to right, the material in the slots are: MW Ruler, 1, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, and E. Coli. The only slot that showed DNA amplification was slot 11, which contained DNA from the unknown bacteria #12.  

PCR and Electrophoresis... Again

Similar to the past 3 weeks or so, I performed electrophoresis and PCR. Slowly but surely, I am making some progress. Last week, I had electrophoresis results that were quite useless. As a reminder, I obtained gels that did not have any visible DNA. After figuring out what the problem was, which was that the SyberGreen-DNA was old/bad, I created another gel and used new SyberGreen-DNA. After obtaining the results, my DNA still wasn’t visible (Figure 1). I then adjusted the amount of post-PCR DNA each slot had from 4 mircoliters to 20 microliters. Finally, this adjustment did show DNA but not for all of my samples (Figure 2). In order to try to get all of the slots to show DNA, instead of using 2 mircoliters of DNA during PCR, I dropped the amount to 1 mircoliter. I will run these samples under electrophoresis next week and hopefully all the slots show DNA!

Figure 1. Electrophoresis results after using new SyberGreen-DNA. The order of the slots, from left to right is as follows: MW Ruler, 1, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 and 14. The post-PCR DNA samples were in PCAT primer set. 

Figure 2. Electrophoresis results after using 20 microliters of post-PCR DNA in each slot. The order of the slots, from left to right is as follows: MW Ruler, 1, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 and 14. The post-PCR DNA samples were in PCAT primer set. 

Continuance of PCR and Electrophoresis

Similar to last week, this week consisted of performing PCR and electrophoresis. However, even though last week’s gel was well done (Figure 1.), it was useless because the PCR primers I used were too concentrated. This resulted in only the primers showing in the gel and not the DNA. In order to dilute the primers from 100X to 1X (concentration), I mixed 1 microliter of a specified primer with 19 microliters of sterile water. Again, this gave me much less concentrated primers and as a result, I should have obtained gels with visible DNA samples. However, that did not occur. Below in Figures 2, 3 and 4 are gels that were ran with post-PCR DNA samples in 1x primers. Figure 2 had Sybergreen-DNA in the gel while Figures 1, 3 and 4 had Sybergreen-DNA mixed with each DNA sample. Sybergreen-DNA is needed because it helps DNA fluorescent, which makes it possible to see. Nonetheless, I still did not have any DNA samples show no matter where the Sybergreen-DNA was. Next week, I will try to figure out what the problem was so that I am able to obtain successful electrophoresis results!

Figure 1. Agarose gel after electrophoresis. In the slots, in order from left to right, are MW Ruler, 1, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 and 14. The mentioned numbers are post-PCR DNA samples with highly (100X) concentrated PCAT-4F-2015 and PCAT-4R-2015 primers from October 5, 2017 PCR run. Sybergreen-DNA was mixed into each DNA sample.

Figure 2. Agarose gel after electrophoresis. In the slots, in order from left to right, are MW Ruler, EC (from October 5, 2017 run), 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 and 13. The mentioned numbers are post-PCR DNA samples with low (1X) concentrated PCAT-4F-2015 and PCAT-4R-2015 primers. Sybergreen-DNA was inserted into the gel.

FIgure 3. Agarose gel after electrophoresis. In the slots, in order from left to right, are MW Ruler, EC (from October 5, 2017 run), 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 and 13. The mentioned numbers are post-PCR DNA samples with low (1X) concentrated PCAT-4F-2015 and PCAT-4R-2015 primers. Sybergreen-DNA was mixed into each DNA sample.

Figure 4. Agarose gel after electrophoresis. In the slots, in order from left to right, are MW Ruler, 14, EC, 1, 3, 4, 5, 6, 7, 8, 9, 10, 11 and 12. The first two samples (14 and EC) are in the PCAT primer set and the ones following these are all in 27F-1492R primers. The mentioned numbers are post-PCR DNA samples with low (1X) concentrated primers. Sybergreen-DNA was mixed into each DNA sample.

Performing Electrophoresis

Last week, the reason I had to wait for my primers to perform PCR (Polymerase Chain Reaction) was because they play an important role. During PCR, these primers target a specific DNA code (Figure 1) and replicate this chain over and over again. This is what happened last week and this week, I used my PCR samples to perform electrophoresis. This process is performed in an agarose gel as electrical currents run through it. At the top, near the DNA slots, it is negatively charged and at the bottom, it is positively charged. Since DNA is negatively charged and considering the fact that opposite charges attract, the DNA is supposed to run towards the positively charged side. Shown below in Figure 2 is the result of my agarose gel after performing electrophoresis.

Figure 1. The primers that will be used to perform PCR and the distinct DNA code each one will target and copy. The values in the 3rd column are the necessary amounts of Tris Hydrochloride needed to be added to each primer before PCR.

Figure 2. The result of an agarose gel after electrophoresis. Looking at the slots at the top, from left to right, the slots contain MW Ruler, 1, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 & 14 (all of the numerical values are post-PCR DNA samples).