Results

DNA Isolation

Samples of purslane, lavender, and water lettuce were collected in order to test the hypothesis that water requirement affects rubisco levels.  DNA was isolated from each sample.   After isolation DNA absorbances were recorded at 260 and 280 nm and used to calculate the concentration of DNA and assess purity of the samples by comparing the ratio at each wavelength (Table 1).

Table 1: Determination of DNA purity and concentration. DNA purity was calculated by comparing the ratio of the abs at 260nm and 280nm. The concentration was found by using the Beer's Law equation.

Real Time PCR

Once DNA was isolated it was used in Real Time PCR to analyze the amount of RBCL genes present in the plant samples.  The RBCL standards did not produce useable results and was thus unable to be used to determine amounts of RBCl in our samples.  Using computer analysis, a melting curve was established for the samples and the control . The melting curve of the samples in which RBCL-fonfana was used (Figure 1) showed little contamination and all had peaks in the same general melting temperature where Rubisco would lie.  However, the no DNA control was contaminated because it too showed a singular peak around the other peaks.  The samples containing RBCL-Savolainen, on the other hand, appeared to have more contamination because there were more peaks than just those of Rubisco (Figure 2).  Thus, analysis was continued using only the RBCL-fonfana samples.  Analysis of these samples was done at cycle 15 when the plate was scanned at 82°C where there was little contamination (Figure 3). Purslane had the highest average C(t) value at 23 cycles while lavender had the lowest value at 16 cycles, followed closely by water lettuce at 17 cycles (Table 2).

Figure 1: Melting curve of samples and control using primer RBCL-fonfana. The large peaks indicate the melting temperature of the samples.

Figure 2: Melting curve of samples and control using primer RBCL-Savolainen. The large peaks indicate the melting temperature of the samples. The extra peaks not clustered together suggest some kind of contamination.

Figure 3: Plate read at cycle 15 at 82°C of samples using RBCL-fonana. Samples were at concentrations of 100 ng.  Dotted line indicates threshold at which C(t) values were determined.

Table 2: C(t) values of samples. Values taken at 82°C at cycle 15.  Concentration of each sample was 100ng.

Agarose Gel Electrophoresis

Gel electrophoresis was used to analyze the results from the PCR to confirm that the PCR product was indeed the RBCL gene.  An image was captured using Biorad software (Figure 4).  The first four lanes contained samples using the rbclf primer.  A band appears near the 500 bp which was predicted for the rbclf primer.  The last 3 lanes are samples using the rbcls primer which shows a band near 200 bp which was also expected for this primer.  The control lanes, 1 and 5, show faint bands which indicate some DNA contamination.  There are also faint smears on all lanes indicating other contamination of some kind.

Figure 4: Agarose Gel of Real Time PCR.  A 2% Agarose gel was run at 100 volts. Lane 1: molecular weight marker, lane 2: rbclf control, lane 3: purslane using rbclf, lane 4: lavender using rbclf, lane 5: water lettuce using rbclf, lane 6: rbcls control, lane 7: purslane using rbcls, and lane 8: lavender using rbcls.

Protein Isolation

Protein was isolated from the leaves of purslane, lavender, and water lettuce.  Absorbances from the protein standards (Table 3) were graphed against protein concentration and fitted with a trend line (Figure 5).  The equation was generated from the graph and was used to calculate protein concentrations for each sample from their absorbances, which in turn were used to determine how much of each sample was needed to perform a Western Blot (Table 4).

Table 3: Absorbance of protein standards at 750 nm.  These standards were used to produce a standard curve from which the unknown protein concentrations from the plant samples were determined.

Figure 5: Standard curve generated from protein standards.  The equation from this curve was used to determine protein concentrations for the samples.

Table 4: Results of protein assay. The concentrations from each sample were calculated from the standard curve equation. These concentrations were used to determine how much of each sample to load on the gels for the Western Blot analysis.

Western Blot

Once the protein was isolated it was run on two polyacrylamide gels.  One was for confirming correct isolation of rubisco, and the other was for Western Blot analysis. The confirmation gel was stained with Comassie Blue (Figure 6) and showed bands for the lane containing a Rubisco standard but not for any of the other samples loaded or the molecular marker.  The Western Blot gel was treated with antibodies for protein detection (Figure 7). The gel revealed bands for both of the lavender samples along with wide, dark bands only at the top of the lanes for the molecular marker, purslane, and water lettuce.  The Rubisco standard didn’t show up at all.  The intensity of the 1:200 dilution of lavender appears to be higher than the 1:20 dilution.

Figure 6: Gel stained with Comassie blue.  Lane 1: molecular weight marker, lane 2: purslane, lane 3: lavender (1:200 dilution), lane 4: lavender (1:20 dilution), lane 5: water lettuce, lane 10: Rubisco standard.  Lanes 6-9 held samples from another group's experiment.  The Rubisco standard is the only lane with any results.

Figure 7: Western Blot.  Lane 1: molecular weight marker, lane 2: purslane, lane 3: lavender (1:200 dilution), lane 4: lavender (1:20 dilution), lane 5: water lettuce, lane 10: Rubisco standard.  Lanes 6-9 held samples from another group's experiment.  This gel was exposed for one hour.

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