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.