Materials and Methods

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Protein Isolation

The protocol for this procedure was followed as noted at Protein Isolation , the following were altered or added to the protocol. 

• When setting up the DC Assay use a Blank, 0.25mg/mL, 0.5mg/mL, 0.75mg/mL, 1.0mg/mL, 1.25mg/mL standards as well as each of the samples x3.

• To make the Reagent SA (the copper containing reagent) 500 µL of SA is needed for each cuvette, therefore 9mL of SA was used.

.          Western Blot

    The protocol for this procedure was followed as noted at Western Blot , the following were altered or added to the protocol. 

• Set-Up - Only 30µL of the protein can be run in each lane, and the concentration much be between 3-30µg, and relatively comparable.  To achieve this, 3.75µL of inorganic lettuce, 1.36µL of organic lettuce, 6.34µL of inorganic onion, and 5.95µL of organic onion in addition to 15µL of 3x loading dye was each added into separate 1mL eppindorf tubes x2 to be added into the wells.

Antibody Detection

    The protocol for this procedure was followed as noted at Antibody Detection , the following were altered or added to the protocol. 

• It may be useful to understand that the nylon membrane is positively charged. Casein, a milk protein, was used as a blocking agent in that it non-specifically bound the positively charged proteins to prevent the antibody from non-specifically binding to the entire membrane. It was desired that the antibody specifically bind to only the Rubisco protein in order to prevent false positives. 

• Note that because the alternative protocol was used in this experiment the primary antibody was diluted to 1:20,000 in 20 mL of TTBS only, specifically 1 µL antibody to 19.99 mL TTBS (nonfat dry milk was NOT used).

• Understand that the washes were performed in order to remove any primary antibody that was non-specifically or inappropriately bound to the membrane. If not removed, antibody that may have been non-specifically bound would skew the results.

• An alternative reaction was used so the secondary antibody goat anti-chicken labeled with a horseradish peroxidase enzyme was diluted to 1:15,000 using 30 mL TTBS/secondary antibody solution, specifically 2 µL goat anti-chicken antibody and 30 mL TTBS (nonfat dry milk was NOT used).

• Only three 5 minutes washes were performed following the first 15 minute wash.

• Overnight exposure was required in this step as exposure for 30 seconds to 2 minutes was not sufficient. 

Shake-Shake-Shake!!!

DNA Extraction

 The protocol for this procedure was followed as noted at DNA Extraction, the following were altered or added to the protocol. 

• Breaking the Plant Cell Wall and Cell Lysis - The frozen plant samples were cut up into fine pieces, and 2g of every sample were weighed out before appropriate reagents were added 

• DNA Extraction - After centrifugation, a 1 mL pipette was used to transfer the upper layer of the liquid into a new 50mL tube. The bottom layer and middle layer were undisturbed during this process. 

• DNA Precipitation - First, 15mL of ice cold isopropanol was added to the samples, allowed to sit for 30 minutes, and then was centrifuged at 2600 RPM for ten minutes. Half of the supernatant was dumped and the rest was pipetted out in order to keep the pellet intact. The pellet was dissolved in 700uL of TE to inhibit DNAses, and 600uL of solution was then transferred to a 2mL microfuge tube. 60uL of 3.0M sodium acetate and 1200uL of ethanol were then added. The solution was centrifuged for 8 minutes, and the pellet was rinsed in 300uL of 70% ethanol.

  The procedure for determining the concentration of  DNA in the samples is outlined in the Real Time PCR section.

Real Time PCR

    The protocol for real time PCR was followed as noted from, Real Time PCR, and the following were altered or added to the protocol  

• 35uL of each DNA sample and 965uL of TE buffer were added to individual

  tubes. A nanodrop was then used to determine the concentration and purity of the

  extracted DNA samples.

• The concentration was then multiplied by the dilution factor to get the original DNA concentration. The dilution factor was 28.5 for all samples except for the inorganic onion. Due to its lower observed DNA concentration more inorganic onion was added so that 100uL of DNA and 900uL of TE buffer were mixed. The observed concentration was then multiplied by the new dilution factor of 10.  

• For all samples another dilution was performed in order to load 100ng of the DNA sample without exceeding more than 8.4uL. The remaining volume was then filled with water. Amounts used for the real time PCR reaction is seen in Table 1.  

• 14 PCR tubes were run. Tubes 1A- 1D were run using Primer B as noted in the protocol. Tube 1E acted as the first control and contained 8.4uL of water instead of DNA, and also used Primer B. Four other tubes were used as duplicates for tubes 1A – 1D. The remaining PCR tubes contained the same DNA as tubes 1A – 1D but used primer A, as well as an 8.4uL control water tube. 

• 11.6uL of master mix containing dNTPs, polymerase, salt, Mg2+, and Sybr green were then added along with the appropriate forward and reverse primers to each reaction tube.  Table 2 illustrates the appropriate amount of master mix and primer needed for the 14 PCR tubes. The sample were then placed in a real time PCR machine for amplification. 

   

Table 1. Shows the adjusted DNA concentrations of each sample after the dilution factor was accounted for. From this value another dilution was performed in order to load 100ng of sample into less than 8.4uL for the PCR reaction. The purity of the DNA collected was also recorded from nanodrop readings.

Table 2. Master Mix and primer volumes used in the 14 PCR tubes.

Agrose Gel

      The protocol for real time PCR was followed as noted from, Agarose Gel, and the following were altered or added to the protocol  

• Casting the gel:  500 µL 50X TAE Buffer, 24.5 mL de-ionized H₂O, and 0.5 g of Agarose powder was added to an Earlenmeyer flask.

• Preparing the samples - 4 µL 6x tracking dye was added to each of four 20 µL DNA RT PCR products (Inorganic Onion, Organic Onion, Inorganic Lettuce, and Organic Lettuce) as well as to the 20 µL RT PCR water blank.

• Loading and running the gel - 4 mL of 50x TAE buffer and 196 mL of de-ionized H₂O  were mixed together in an Earlenmeyer flask and used to fill the buffer reservoir and cover the gel.

• Each designated well received 15 µL of DNA sample or blank; 10 µL of molecular ladder was added to the central well.

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BIO 480

James Madison University