Methods
For this experiment, S1 designates green Acer saccharum, S2 designates yellow Acer saccharum, S3 designates green Fraxinus pennsylvanic, and S4 designates yellow Fraxinus pennsylvanic.
Protein was isolated from four plant samples by grinding one gram of finely cut leaf sample with QB Buffer. This was done individually for each sample. Each sample was given a label (S1, S2, S3, S4). Samples were then centrifuged at 4ºC for fifteen minutes. A DC Assay was set up using six standards (blank, 0.125 mg/ml, 0.25 mg/ml, 0.5 mg/ml, 0.75 mg/ml, 1.0 mg/ml) and eight samples (10μl and 5μl of S1 and S2, 1μl and diluted 1μl of S3 and S4). To each of these cuvettes 500μl of solution A, 10μl of solution S, and 4ml of solution B. They were allowed to sit for about 15 minutes before being tested in a spectrophotometer. Absorbencies were recorded and protein samples were stored at -80ºC.
Four to ten leaves from each sample were ground with 20ml of CTAB reagent. An alternative method was used at step thirteen. Instead the pellet was spun for two minutes to extract the last of the supernatant. Samples were stored in the refrigerator for a week. Determination of the DNA concentration was carried out after that week.
Nine reactions were set up. These were 100ng of S1, S2, S3, and S4, 50ng of S1, S2, S3, and S4, and the blank. Primer set A was used (rbcl2f and RBCL-fontana) to get a fragment of about 500bp. The reaction was left to run over night.
Four of the wells were filled with 10μl of each PCR reaction mixed with 2μl of 6x loading dye. These PCR reactions consisted of the four 100ng samples. A 1Kb Plus ladder was used.
Two pre-made acryl amide gels were used. Gels were shared between two groups and both gels were made identical to each other. Four wells were used on each gel that contained 15µl of 3x sample loading buffer and one of the following: 30µl of S1, 30µl of S2, 25µl of S3, or 25µl of S4. The first gel was stained over night and photographed. The second gel was used in the Western Blot. The next day, the membrane from the Western blot was washed in a buffer solution and then stored in a blocking solution (25ml TTBS, 2.5g non-fat dry milk) for one week.
The membrane was washed using an alternative protocol which used 15ml of TTBS and 3µl of Chicken anti RBCL. The second wash was done using 15ml of TTBS and 1µl of rabbit anti-chicken with horse radish peroxidase. The procedure for the light reaction was followed.