RESULTS

 

DNA Analysis

By quantifying and analyzing DNA integrity from our samples we can determine a relative quantity of the RBCL gene in our 2 species of flowering plants treated with differing light intensities.  Spectrophotometer analysis of our DNA extractions showed that both Impatiens samples were relatively pure (1.8-2.2) but contained less DNA than both Nirvana  which had a relatively low purity
(Table 1.) 

 

Table 1. Spectrophotometer analysis of DNA extractions from respective samples.  Concentrations determined using an absorbance coefficient of 50 ug/uL is equal to one O.D. High purity is considered to be above 1.8 which both Impatiens sp. were over and both Nirvana sp.  were under. High concentrations of both Nirvana sp.  treatments were observed.
  Impatiens sp. Sun Impatiens sp.  Shade Nirvana
Sun		 Nirvana
Shade
    A260 0.174 0.132 0.22 0.219
    A280 0.09 0.071 0.275 0.26
    Pur 260/280 1.9 1.9 0.8 0.8
    Concentration,
    ug/ uL		 0.248 0.189 0.315 0.313

 

 

 

 

 


 

Figure 4. Agarose gel of 50 ng sample RT-PCR products . 2% agarose gel made with 1XTAE buffer, ran with a 1kb ladder, primer control (C) and stained with ethidium bromide. Lane designations: (IS)-Impatiens Sun, (ID)- Impatiens Shade, (NS)-Nirvana Sun, (ND)-Nirvana Shade.

 

 

 

Real Time polymerase chain reaction of DNA samples was used to assess the quantity of RBCL gene.   For simplicity sake we only included our data for one of our 100 ng duplicates. Cycle numbers were determined at the point the slope crossed the threshold line (Figure 1)Tm for the primers was approximately 79ºC and for all samples the average was 82.7°C (Figure 2) . Temperature of annealing of the rbcl2F primers was determined by estimating the middle of the two peaks in Figure 2, giving a value of 81.5°C.  For comparitive analysis, synthetic RBCL standards of known concentration were also subjected to RT-PCR analysis (1024pg, 512pg, 256pg, 128pg, 64pg, and 0pg).  In order to get the best comparison to determine the relative DNA concentrations in our samples, only choice standard concentrations were used. (Figure 3 a-c).   Agarose gel electrophoresis of our RT-PCR products shows bands for each lane at approximately the same weight, ~300bp, along with primer dimers at the very bottom of the gel (Figure 4).

                                                                              
Sample Ct
Impatiens Sun 2.81
Impatiens Shade 12.79
Nirvana Sun 9.14
Nirvana Shade 6.76
Blank 24.37





 


                                    
    



Figure 1.  Relationship between log of fluorescence of SYBR Green Dye and threshold cycle of various samples containing an estimated 100 ng of DNA. Plate reads were taken every 0.2C from 65C to 95C.  Point at which slope of line crosses "Threshold", that cycle #,  is indicative of relative abundance of template.

 

 

 

                                               

 

 

 



 

 

Sample Tm(°C)
Impatiens Sun 82.8
Impatiens Shade 83.0
Nirvana Sun 82.4
Nirvana Shade 82.4
Blank (Primers) 79.6

 

 

 








Figure 2.  Melting curve for rbcl2 primers and same 100ng estimated DNA from various samples. Derivative of slope is graphed alongside melting curve.  Slope of melt curve is indicative of Tm between primers and the sample. 

       

 

 

                

 

 

 

 

 

 

 

 

Figure 3. Melting curve, quantification curve, Ct, and Tm for choice RBCL gene standards. Standards that represent data flow well were chosen to display in each figure. (a) Melt temp analysis of standards with derivative of slope equal to Tm. (b) Quantification curves for same choice standards. (c) Table of standards used with color key and Ct and Tm values.

 

 

Protein Analysis

In order to quantify the amount total protein in each of our isolated samples we use spectrophotometer analysis alongside protein standards. Absorbance were measured by the blue hue produced from DC Assay (Figure 6a)  The relationship between the concentrations of our protein standards and their absorbance showed a high correlation coefficient, R2 = 0.9959. Exact concentrations of the total protein in our samples was determined using the equation generated from the best fit line (Figure 5 b-c)


 

 

 

 

 

 

 

Standard

 Ct Tm(°C)
256pg 13.29

    83.8
128pg 15.49 82.4
64pg 19.43 85.8
32pg 23.327 85.2
16pg 23.72 85.6
0pg 24.37 79.6

 

 

 

 

 
 


 
 Sample A750 Protein Concentration, ug/uL
Impatiens Sun 0.285 2.03
Impatiens  Shade -0.018 0.315
Nirvana Sun -0.054 0.111
Nirvana Shade 0.053 0.717

 

 


Figure 5. Relationship between protein concentration and 750 of protein standards. (a) Image of isolated protein samples treated with DC protein assay (BioRad). The characteristic blue color is produced from the reduction of the Folin-Ciocatlteu reagent and its intensity id dependent upon the amount of protein. (b) Protein standard data plotted and equation generated with Microsoft Excel and used to determine the protein concentrations of our samples. Table depicting absorbance values and protein concentrations of samples calculated from best fit line generated in (b) (b)                                                                                                                   

                              

 

 

 

 

To observe RBCL expression amongst our samples we performed SDS-PAGE followed by a western blot.  The only detectable results can be observed through antibody visualization in the western blot.  Protein samples from Nirvana sun yielded 3 bands; one at ~2000kD, and 2 below 650kD, and Nirvana shade yielded 2 bands; one at ~2000kD and 1 below 650kD.  The positive control yielded a light band.  The lower band observed for Nirvana Sun have a relative darker intensity than the Nirvana Shade bands.  Weights of bands were approximated from the barely discernable ladder observed on the SDS-PAGE. (Figure 6 a-c)



 

 

 

 

 

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