EUREKA!!! (almost)

The PCR process was successful! Well, sort of… As is seemingly the norm for me, my results introduced some new questions. I’ll get to that in a moment. First and foremost, when electrophoresis was conducted using my PCR results, there were evident bands of DNA that traveled in the gel. This means that even though there was sheering evident in my extraction method, there was a viable product that yielded suitable fragments of DNA that could be amplified through PCR.

The chloroplast primers I used were not suitable for purposes of differentiating between the individual test subjects of my research. These primers are very general in nature and were just used to see if the extraction results contained amplifiable DNA. This is the Eureka part. The new questions arise from where the bands occurred on the gels.

PCR was conducted with the results from three different extractions: the 1^st was the extraction that appeared to have a massive amount of DNA with heavy shearing, the 2^nd was the same protocol but done in a linear manner (in the 1^st the protocol the run was broken up due to time constraints, and this may have caused shearing), and the 3^rd was the same as 2^nd run, with the addition of using improvised wide bore pipette tips (again to try to reduce shearing). Each sample went through PCR using 3 different concentrations of each extraction for a total of 9 PCR reactions. The banding appeared in the wells containing the 1μL and 2μL extraction concentrations. There was no banding/travel exhibited in the 3μL concentrations. To relay a clearer version of what I observed in the 3 gels, I reran in a new gel omitting the 3μL concentrations. Below is the image of that gel.

As wonderful as it was to observe a discernable band of PCR results travel in a gel, my elation was cut when I noticed where the travel occurred. Evident bands appeared in two different extractions at two different concentrations! Even though I was extremely meticulous in labeling, handling and loading the samples, it is my hope that I mislabeled or misloaded one of the samples. I cannot come up with a viable explanation for these results – it just does not make any sense.

It appears as though next week I will give it another run…