Results
Protein Isolation and Analysis
Rubisco extraction from the root, stem and leaf was the first step in determining Rubisco variance of the elephant ear plant. The procedure can be found in the methods section. The DC protein assay (BioRad) was used to determine the concentrations by plotting five standards with known concentrations on a graph (Figure 1). The absorbance of each sample at 750nm was compared to a standard curve in order to determine the concentration. This was done by plugging the absorbencies into the trend line equation. A spectrophotometer measured absorbencies at 750nm (Table 1). These concentrations were used to calculate the amount of each sample that was needed to load on the acrylamide gel for the Western Blot technique. These values can be seen in Table 2.
The samples were then run using polyacrylamide gel electrophoresis (PAGE), which separates proteins based on molecular weight. Using electrophoresis, the proteins from the gel were transferred to a membrane. The proteins remained in the same position when transferred. Once on the transferring was complete, chicken antibodies were used to detect the Rubisco protein on the membrane and goat antibodies were used to detect the chicken anti bodies. The enzyme called horseradish peroxidase breaks down the substrate and caused emission of light, which was detected by a BioRad Chemilumenescent camera that took a photo of the membrane as seen in Figure 2. A molecular weight marker that was added to the gel was used to determine the position of Rubisco, which is 53 kb. The membrane displayed a heavy band at approximately 53 kb for the lane containing the leaf sample, while the stem an extremely faint band and the root sample had no band displayed by Figure 2. The stained acrylamide gel also displayed bands at this molecular weight clearly for the stem and leaf samples, but smeared for the root sample, note Figure 3.
DNA Isolation and Analysis
DNA was extracted from each region of the plant using the protocol from the methods. The dilution used to calculate the concentration for PCR was 200μL of sample/800μL of water. This dilution was necessary to achieve an absorbance between 0.1 and 1.0 because, within this range, the measurement is the most accurate. The absorbencies, concentrations, and purity readings are found in Table 3. The initial absorbencies at 260 nm were used to determine the concentrations of the samples. Absorbencies of 260 nm and 280 nm were used to calculate purity of the sample. Only the leaf sample had a good purity reading of 1.97, since it was between 1.8 and 2.0. However, the root and stem samples were outside of that range indicating a large amount of unwanted proteins.
The Real-Time PCR was performed with the use of two different primers, A and C. When looking at the results from the Real-Time PCR, the C(t) values for all of the samples are fairly similar, with only a difference of 6 cycles between the least and greatest amounts of Rubisco DNA expression. It actually shows the stem with the highest expression with a C(t) value of approximately 22, followed by the leaf, and then the root. (Figure 5 ) The melting curve displays what should be found, with the negative control melting before the rest of the samples. (Figure 6 ) Primer A’s melting curve showed better results with the negative melting off; however, primer C’s curve has everything melting at the same time. Primer C’s curve was run twice (trial1 and trial 2) and showed better results the second time, with the negative melting at 75.5 and 80 degrees Celsius, while the first of the samples does not melt until 81.5 degrees Celsius. Thus, the temperature from the first graph is probably taken around 80 degrees Celsius, because it is after the negatives are melted but before 50% of the samples are melted.
The samples from the Real-Time PCR were also run on a gel and produced very similar results as seen in Figure 4. The PCR is run with four all of our samples, a negative, and molecular weight marker. The marker is what is used as a scale, to measure what is in the samples. The gel showed that all of our samples including the negative had some protein in it. The Rubisco sample showed bands at 100 bp and 200 bp, which is what all of our samples showed including our negative. The leaf and stem measurements are much bolder than that of the roots.
Introduction Methods Discussion
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