R u b i s c o
RESULTS
Determining stock protein concentration
To determine the stock protein concentration from the isolated samples (2, 4,6, and 8 weeks of Arabidopsis thaliana), a Lowery-Based protein assay was performed for each sample and the absorbance at 750nm in Table 1. Absorbance were also obtained from protein standards range from 0.125 mg/ml to 1 mg/ml. The protein standards were plotted and fitted with a trendline. An equation and R2 value was obtained for that trendline, which can be seen in Figure 1.
.
Figure 1. Scatter plot showing the standard curve. Scatter plot showing the standard samples and fitted with a linear trendline. The absorbance of each standard sample was taken at 750nm. An equation and R2 value was found for the trendline and is displayed on the graph. The R-squared value was 0.95, showing that there is approximately a 95% chance that our data is not random.
|
Sample |
Dilution |
Abs |
Concentration (ug/mL) |
Concentration (ug/ul) |
Volume to load in uL of each sample |
|
4 wks |
30/70 |
0.494 |
n/a 9.47 18.2 |
0.0138
|
30 |
|
95/5 |
0.134 |
||||
|
99/1 |
0.052 |
||||
|
6 wks |
30/70 |
0.537 |
n/a 10.8 23.4 |
0.0171
|
24.3 |
|
95/5 |
0.153 |
||||
|
99/1 |
0.067 |
||||
|
8 wks |
30/70 |
0.333 |
38.6 8.01 14.7 |
0.0204
|
20.3 |
|
95/5 |
0.113 |
||||
|
99/1 |
0.042 |
||||
|
10 wks |
30/70 |
0.235 |
27.3 3.44 12.3 |
0.0143
|
28.9 |
|
95/5 |
0.047 |
||||
|
99/1 |
0.035 |
Table 1. Stock protein concentration and volume ( mL) to use for Western Blot Gel. The concentration for each dilution was calculated based on the absorbance at 750 nm and the equation fitted to the standard samples shown in Figure 1. The concentration in mg/mL was then converted to mg/ mL. The equation for the trendline was used to caluculate the volume need to load 30 ng of protein into the Western Blot gel in mL
Figure 2. Concentrations of protein extraction samples. The concentrations were determined from Table 1. The sample is indicated below the x-axis and the concentration of protein is shown on the y-axis
Quantitative Polymerase Chain Reaction (qPCR) Results
The DNA was extracted from the plant samples taken from 4, 6, 8 and 10 week old plants. The same amount of DNA was loaded in each sample. Two separate sets of Primer were used for our qPCR Reaction. The primer set rbcl2f, which contains forward primer Reiseberg and reverse primer, RBCL-fonana cuts a section of DNA of approximately 500bp. The second primer set rbcl2F contains the forward primer Rbcl Reiseberg and reverse primer RBCL-savolainen cuts a section of DNA of approximately 200bp. Figures 3 and 4 show the fluorescence of each of the samples with respect to the cycle. The more fluorescence is an indication of more DNA. For both primer sets, weeks 4 and 6 were amplified faster than weeks 8 and 10. Ct values were determined and show which cycle each sample crosses a given point of fluorescence and are displayed in units of cycle number (cycle #). This is a measure of the amount of DNA in the samples. The Ct values for our data using each of the primer sets is displayed in Table 2.
Figure 3. Quantitative PCR Quantification DNA using Primer rbcl2f. Amplification of the DNA target sequence in each sample is shown on the graph as a measure of fluorescence versus cycle. The x-axis shows the cycle number and the y-axis shows the amount of fluorescence for each sample. The no template control is shown as the red line and labeled "NTC." The color indication with the Ct values for the samples is shown in Table 2.
Figure 4. Quantitative PCR Quantification DNA using Primer rbcl2F. Amplification of the DNA target sequence in each sample is shown on the graph as a measure of fluorescence versus cycle. The x-axis shows the cycle number and the y-axis shows the amount of fluorescence for each sample. The no template control is shown as the red line and labeled "NTC." The color indication with the Ct values for the samples is shown in Table 2.
Melting curve was obtained from the RT-PCR shown in Figure 5 and 6 with primer sets rbcl2f and rbcl2F respectively. These graphs allow us to determine the melting point of each of the samples, which gives an indication of purity and consistency between samples.
Figure 5. Melting curve of primer sample set rbcl2f. The melting points for the primer set rbcl2f showed that all samples melted at approximately the same temperature. The NTC sample had two melting points, one before the rest of the samples and one at approximately the same temperature as the other samples.
Figure 6. Melting curve of primer sample set rbcl2F. The graph shows that there were several different melting points for our samples Overall, the NTC had a lower melting point then the samples, which were grouped around the same area. This graph indicated much more noise when compared to that of the other primer set displayed in Figure 5.
|
SAMPLE |
Ct Value (cycle #) |
|
rbcl2f |
|
|
4 weeks |
16.461 |
|
6 weeks |
15.932 |
|
8 weeks |
18.888 |
|
10 weeks |
18.359 |
|
NTC |
0 |
|
rbcl2F |
|
|
4 weeks |
16.638 |
|
6 weeks |
16.787 |
|
8 weeks |
18.913 |
|
10 weeks |
18.453 |
|
NTC |
0 |
Table 2. Color coded samples for qPCR with primer sets and Ct values (cycle #). Each sample run in the qPCR reaction is displayed in the same color used for that sample on Figures 4-6. The Ct values for each of the samples are displayed.
To show the display the qPCR data in a more comparative fashion a graph of all Ct values is displayed in Figure 7. Due to the fact that this experiment was conducted only once no statistical analysis could be performed to determine the significance of our results. Therefore the following figure merely shows trends in data obtained at one time and not conclusive results of the experiment.
Figure 7. This graph illustrates the Ct value, or cycle number that each sample crossed a given threshold. Samples with primer set rbcl2f are shown in purple and samples from primer set rbcl2F are shown in pink, as displayed in the legend to the right of the graph. There is a slight trend shown in that all the rbcl2f samples were had lower Ct values than the rbcl2F samples. Additionally, the 4 and 6 week samples had lower Ct values than the 8 and 10 week samples, with eight weeks being the highest for both primer sets.
Electrophoresis using the samples from the qPCR reaction were run on a 2% Agarose Gel to verify qPCR results. The samples were run on the agarose gel for approximately 40 minutes at 110Volts. The gel was then stained with ethedium bromide and exposed to UV light to generate the image shown below in Figure 8. The DNA fragment came out to be the same size as the negative primer controls shown in figure 8.
Figure 8. Electrophoresis on a 2% agarose gel of qPCR Samples. Lanes 1-5 are samples from the rbcl2f primer set, for the NTC, 4, 6, 8 and 10 weeks respectively. All these samples, including the NTC display bands at approximately 500 bp. Lane 6 contained a 1 kb molecular marker, which is labeled above in white. Lanes 6-10 are samples from the rbcl2F primer set and are the NTC, 4, 6, 8, and 10 week samples respectively. Bands appeared in all lanes, including the NTC at 200 bp.
SDS PAGE Gel
Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS PAGE) was performed to separate proteins from a crude leaf extract based on size. Approximately 4 mg of protein was loaded in each sample. The acrylamide gel was stained with a Comassie blue stain and photographed under white light to generate the image shown in Figure 9.
Figure 9. SDS PAGE Gel of curde leaf extracts. Lane one is a molecular marker, which is unlabeled because the marker did not show all bands. Lanes 2-5 are the crude leaf extracts for samples at 4, 6, 8, and 10 weeks. No distinct bands can be seen. The sample lanes show a distinctive "V" protein pattern.
Western Blot and a Commassie Blue Stain were performed for quantitation of the protein samples. (The amount of protein loaded on the western gel was shown in Table1. However, none of these gels had showed any possible trace of protein band. (Data not included due to no result outcome.