Results

Figure 1. Acer saccharum. Courtesy of

http://www.forestry.auburn.edu/samuelson/dendrology-

/images/aceraceae/sgr_maple%20L2.jpg

 Figure 2. Fraxinus pennsylvanic Courtesy

http://www.forestry.auburn.edu/samuelson/-

dendrology/images/oleaceae/gr_ash_L2.jpg

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Protein Analysis: 

The green and yellow samples from the Acer saccharum were labeled S1 and S2, respectively.  The samples of the green and yellow leaves from the Fraxinus pennsylvanic were labeled S3 and S4, respectively.  A DC protein Assay was used to make a standard protein curve. (Figure 3).  With the equation of the curve’s trendline, each sample's concentration was determined from their absorbencies at 750nm.     

     Figure 3. Graph showing the protein concentrations of the protein standards when the absorbance was at 750 nanometers.

The first time the sample’s absorbencies were observed, the proteins were diluted 1ug/1ml.  These each produced absorbance readings outside the range of the standard protein curve (Figure 3).  A second trial was preformed in which each stock sample was diluted five fold and ten fold.  The absorbencies obtained from these trials were used, in conjunction with the standard curve equation and the sample’s given dilution factor, to determine the concentration of the stock protein sample (Table 1). 

                                                       Table 1. shows the protein concentrations of the plant samples at 750nm.

The stock concentrations shown in table 1 were used to determine the amount of protein necessary to run a Comassie blue stained polyacrylamide gel.  Each lane was loaded with about 30ug of the protein.  From the photograph of the gel, shown in figure 4, it is evident that there was no visible protein in any of the lanes of interest. The molecular marker (lane 4) was used to determine the location of the large subunit of the Rubisco protein, which was expected to show up around the bottom violet band, in lane 5.

Figure 4. Acrylamide gel with protein samples.  The first three lanes belong to another group. 

Lane 4 is the molecular weight marker. Lane 5 is S1, lane 6 is S2, lane 7 is S3, and lane 8 is S4.

After following the Western Transfer Blot Protocol, the western transfer membrane was visualized with a BioRAD Chemilumenescent camara (Figure 5).  As expected, the pattern seen on this gel was identical to that of the acrylamide gel (Figure4). With the exception of a faint smudge across the first lanes 5, 6 and 7, the membrane shows no distinct sign of protein.  The smudge appears to be at the right height for the Rubsico protein, however it is not definitive enough to show the presence of protein.       

Figure5.  The westen transfer membrane after addition of antibodies.   The first three lanes contain another groups protein samples. 

Lane 4 is the molecular weight marker. Lane 5 is S1, lane 6 is S2, lane 7 is S3, and lane 8 is S4. 

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DNA Analysis:                                                                             

The DNA of each of the four samples was extracted.  By diluting the isolated samples by 50 fold, the concentration of DNA could be determined using its absorbance from the spectrophotometer at 260nm.  The 260nm level is significant because the nitrogenous bases absorb light at this frequency.  Knowing that the aeromatic portion of amino acids absorb light around 280nm, each samples purity was determined by the ratio ABS(260)/ABS(280).  A ratio of 1.8 means that the sample is very pure.  These results are shown in table 2, and indicate that the samples of isolated DNA were not pure.

Table 2 shows the concentrations of DNA in the samples at 260nm and 280nm.  Samples S1, S2,

and S3 had a dilution factor of 40 and sample S4 had a dilution factor of 200.

In order to verify that that the gene for Rubisco was present in the plant samples at equal concentrations, a real time PCR was run so that the data could be easily quantified.  A total of 16 individual reactions were run.  Eight of the reactions consisted of sample S1-S4 being run at different dilutions with Primer A.  Each of the samples that were run with primer A were tested once with 100ng of DNA, and once with 50ng or DNA.  The other eight reactions consisted of samples S1-S4 being tested twice at the dilutions 100ng and 50ng, with primer C.  The results for the reactions run with samples having 100ng of DNA are shown in Figure 6.  Both show that no C(t) value was able to be determined from the reactions preformed.  The data for the PCR reactions run with 50ng show the similar results, as seen in Figure 7.  Without being able to use the PCR  results to determine the C(t) values, it is impossible to determine which sample is being amplified first.  In other words, this data gives us no insight in to the relative DNA concentrations of the samples. 

Table 3 shows the amounts of DNA used in the real time PCR reaction.

Figure 8.  Gel of real time PCR products.  Lane 1 is sample S1. Lane 2 is sample S2. Lane 3 is sample S3.

Lane 4 is sample S4. Lane 5 is the molecular weight marker.  There was no distinctive DNA in the first four lanes.

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