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Results

I.  Sample Collection

The samples of spinach used in this experiment were bought from a Kroger Grocery Store in Harrisonburg, Virginia.  They were stored unopened for three days after purchase in a personal refrigerator.  Figure 1 shows the sources of all samples used and Table 1 presents descriptive information about the type and quality of each sample.   Three of the sources were produced by Dole while only one was produced by Fresh Express.  The samples differed in there "Best By" dates and exhibited different degrees of wilting. 

Figure 1.  Photographs of the sources from which samples were taken.  All sources were bought from a Kroger Grocery store at the same time and store in a personal refrigerator for three days prior to sample collection.

Table 1.  Descriptive information of sources and observations of samples upon collection.  All sources had different expiration date, with the greatest difference among any being 8 days. The color and amount of wilting differed among all sample types. 

II.  Protein Quantification

Two different dilutions of our samples were analyzed spectroscopically by obtaining absorbance readings at 700nm.  Figure 2 shows that as the concentration of protein increase, so does absorbance.  This direct relationship is operationalized in Beer's Law : A=ebc, where c, stands for the concentration.  Because we don't have an extinction coefficient, e, for protein in general, we plotted the values given in Table 2 and added a trendline as show in Figure 2.  The equation of this line was use to determine the concentrations of our samples, displayed in Table 3.  It is interesting to note that the FBO samples in both dilutions showed markedly higher absorbance readings. 

Figure 2.  Standard curve for the DC Assay is plotted with absorbance measurements obtained for solutions of known protein concentration at 595nm.  These concentrations and their absorbance’s at 750nm are shown in Table 1.  The equation of the trend line: y = 0.1841x + 0.0614 was used to calculate the concentrations of protein in our samples, shown in Table 2. The R² value given for this line reveals a moderate amount of scatter around this line.

Table 1.  Absorbance values for solutions with known concentrations of protein used in plotting the standard curve in Figure 2.

Table 3. Concentrations of protein samples as determined in the DC Assay using the standard curve plotted in Figure 1. The single starred samples (*) contained 1µL of the original in 99µL QB Buffer in the cuvette.  The double starred samples (**)  contained 5µL of the original in 95µL QB Buffer in the cuvette.  It is important to note that while we attempted this experiment with a less diluted sample of each spinach type (30µL  of sample to 70 µL  of QB buffer) the absorbance reading for these samples were erroneous, giving negative numbers.  

III.  DNA Extraction

After DNA extraction, each sample's concentration was determined using the absorbencies obtained from a spectrophotometer.  Theoretically, the A260 measurement can be used for a measure of DNA content while the A280 measurements can be used for that of DNA and protein because the aromatic amino acids absorb in this range.  To determine the purity of each sample, the absorbance at 260 was divided by that at 280 nm.  A pure sample typically has an A260/A280 ratio between 1.8 and 2.0.  The absorbencies, and calculated purity, are shown in Table 4.  It is clear that our sample lie above this range and therefore our purity is compromised. 

Table 4.  Absorbances of each sample at A260 and A280.  The A260/A280 ratio is used to determine purity.  A ratio between 1.8 and 2.0 is considered a pure sample.

IV.  Real Time PCR

Real Time PCR was performed to determine the amount of rubisco-coding genes found in each sample of DNA obtained in step 2.  Primer C was used to amplify the rubisco gene in the polymerase chain reaction.   
 

Figure 3.  Description of sample used in each lane of RT-PCR.  PC indicates that Primer C was used.  Concentrations of 50 and 100 ug of DNA from each sample were used. 

Figure 4.  The cycle number vs. Fluorescence of DNA in step 11 of Real-Time PCR.  In step 11, the temperature is 80°C.  The sample information can be acquired by matching the color from the figure to the legend in Figure 2. 

Figure 5.  Real-Time PCR of all samples using Primer C with an H2O blank.  This is the melting curve graph for the Polymerase Chain Reaction.  The sample information can be acquired by matching the color from the figure to the legend in Figure 2. 

Table 5.  Real-Time PCR data for 50 and 100 ng DNA samples using primer C.  The C(t) value represents the threshold count for each sample to determine which has the highest DNA concentration.  PC indicates that Primer C was used.  FBO indicates that Fresh Expressed Boxed Organic spinach was used.  DOS indicates that Dole Organic Spinach was used.  Dole Conventional Spinach was abbreviated as DCS, and DBC indicated that Dole Boxed Conventional spinach was used. 

V.  Agarose Gel of Real-Time PCR

PCR samples were run on agarose gel to separate DNA that was made in the PCR using Primer C.  The samples in the gel were the same samples used in the PCR because the goal was to check if the band was the right size.  In an 8-well agarose gel, a marker, a PCR blank, and an agarose gel blank were used, as well as 5 samples from our DNA.  Of the 5 DNA samples, 4 were from the 50 ug concentrated DNA of each spinach sample, and 1 was from the 100 ug concentrated DNA of DBC spinach.  By showing a series of compounds with known molecular weights, the ladder shown in lane 5 allowed us confirm that our bands did contain Rubisco.

Figure 6.  Agarose gel of PCR products.  Lanes 1 and 2 contain agarose gel blank and PCR blank, respectively.  Lanes 3, 4, 6, and 7 contain 50 ug DNA concentrations of FBO, DCS, DOS, and DBC, respectively.  Lane 5 contains the 1 kb plus molecular weight marker.  Lane 8 contains 100 ug DNA concentration of DBC. 

VI.  Western Blot

Two SDS-PAGE Gels were used to separate the proteins.  One of the gels was stained using Comassi Blue overnight, and a picture of the gel was taken.  The second gel was transferred onto a membrane via electrophoresis.  For all four spinach samples, the volume needed to get 30 ug of DNA was calculated, and used for both gels.  Lane 1 contained 4.3 ul of DCS DNA, lane 2 contained 4.2 ul DBC DNA, lane 3 contained 4.0 ul of FBO DNA, and lane 4 contained 4.3 of DOS DNA.  Lane 5 contained the 1 kb plus molecular weight marker, and lane 10 contained loading dye.  Lanes 6-9 were used by the group that was paired with us.  This method of preparation was used for both gels.  There is an overload of dark bands on both the gel and the membrane which confirm that we did have the large Rubisco subunit in our samples.  However, given the similarity of our samples, the thickness of all the bands made it impossible to draw and conclusions about the relative amount of rubisco in each sample.

Figure 7.  SDS-PAGE acrylamide gel after staining with Comassi blue dye.  Lane 10 contains the loading dye, and lane 5 contains the molecular weight marker.  There are bands visible in lanes 1, 2, 3, and 4. 

Figure 8.  Western blot membrane. The bands present in lanes 1, 2, 3, and 4 indicate that Rubisco has been successfully located.  There is not any indication of a lot of nonspecific binding. 

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