James Madison University
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Discussion of Results
We chose to analyze chrysanthemums because of the variety of plants available at different growth stages when we began the project. Our hypothesis was that in the leaves and stems of a plant that is just beginning to bud, a low amount of RBCL DNA will be observed; in a plant that is growing some flowers, which are enlarging, levels of the protein and DNA will be great, and in a plant which has already bloomed and is beginning to degrade, levels of RBCL DNA will be about half the maximum.
We attempted to isolate the RBCL protein and measure the concentration via spectrophotometric readings at 750 nm. A standard protein curve was used to determine the concentrations of our samples from the absorbance readings (Table 1). We then used the concentrations found to determine the volume needed to load 30 µg of each sample on a polyacrylamide gel (Figure 4); however, no bands were present after electrophoresis and staining. This could be because the absorbance readings at 750 nm were not really optimized for the RBCL subunit, and therefore the concentrations that we calculated may actually describe other protein concentrations. Also, all of our calculated protein concentrations were toward one extreme end of the standard curve, which may have contributed to the miscalculation. This error could be avoided next time by creating a standard curve with a wider range of standard dilutions. A Western blot was also performed using a second gel, and no bands were observed. This was expected since we also saw no bands on the analogous polyacrylamide gel which was stained. The RBCL-positive control barely showed any bands, indicating that our technique for the Western blot may have been inadequate.
The amount of RBCL DNA in each of the three plants was evaluated by PCR using forward and reverse primers that are specific to the RBCL gene sequence. Unfortunately, we were unable to make any conclusions from the agarose gel run after the real-time PCR experiment since no bands were clearly observed on the gel (Figure 11). The threshold cycle and melting curve data for our samples was also inconclusive (Figures 7, 8, 9, 10). On the melting curve graphs, no plant sample DNA showed a distinct peak at melting temperature. On the quantitation graphs for determining threshold cycle, none of our samples crossed the threshold value, which could be a result of primer/RBCL DNA compatibility. The no DNA control It is possible that during DNA isolation some nucleases were introduced to our samples and the DNA was degraded before it could even be used as a template in PCR. It is also a possibility that the primers we chose were not selective for our plant species’ RBCL DNA.
We conclude that our hypothesis cannot be accepted or rejected based upon the inconclusive results obtained. If we had more time, we would start from the protein samples previously isolated and attempt to quantify the protein concentration of our samples and the standards using several different wavelengths of light to determine a new absorbance to concentration relationship. Then the SDS-PAGE could be reattempted, hopefully using a greater mass of protein, and if bands did indeed result, a new Western blot would also be performed. As for the DNA, we would most likely want to re-isolate DNA from the samples, and possibly perform an additional mini-prep to purify the DNA before going on to the real-time PCR experiment.