Methods and Materials
The two species chosen to compare the effect of farming methods were, Collard greens (Brassic Oleracea) and Parsley (Petroselinium Crispum). Four independent tissue variables were used: Organic Collard Green, Non-organic Collard Greens, Organic Parsley, Non-organic Parsley. Plants were chosen due to their availability and were purchased at Martin’s food store in its respective organic and non-organic sections of the produce department. For both the parsley and the collared greens, tissue was selected from the leafy area of both plants and not from the stem area.
Protein Isolation
The goal of this procedure is to lyses the cells and extract protein for stable storage. Cold temperatures and proteases inhibitors were used to minimize protein degradation. A Lowery- Assay was preformed to determine protein concentration. This technique detects the reduction of the Folin-Ciocalteu, which produces a blue product, by measuring absorbance at 750nm. Dr. Rife's procedure was modified to isolate 120 ug of protein. Protocol used was from Dr. Rife: Protein Isolation.
Western Blot
Two gels were created and ran for the Western Blot protocol. Each gel was loaded with 60 ug of protein. One gel was specifically an SDS-PAGE gel to separate proteins by size and their surface charge and give indication that isolation had worked properly. This gel was stained with Coomassie Blue and a picture was taken of it for analysis. The second gel run was also an SDS-PAGE gel but its purpose was to be used for the Western Blot. The gel was transferred to a membrane and washed and placed in blocking solution. The protocol used was from Dr. Rife: Western Blot.
Next, the membrane was exposed to a primary and secondary antibody to hybridize to the membrane. The membrane is washed repeatedly and then stained with luminol/enhancer and peroxide buffer to induce a light reaction. The alternate protocol was followed for analysis of the Western Blot. The membrane was left to expose and a BioRAD Chemilumenescent camera was used to take a picture. The antibodies used were grown in chicken. The protocol used was from Dr. Rife: Antibody Detection.
DNA Isolation
This procedure was designed to isolate pure DNA from the plant tissues via a chloroform/alcohol method. Concentration of the samples was measured on ND-1000 spectrophotometer (Nanodrop). Protocol used was modified from Doyle & Doyle (Photochemical Bulletin, 1987) and Clevinger & Panero (American Journal of Botany, 2000): Genomic DNA Purification from Plant Sources.
Real Time PCR
The purpose of real time PCR is to determine the number of RBCL copies present in the sample. Real time PCR offers the advantage of being able to visualize double stranded DNA products as they are being synthesis in the reaction. Protocol uses was from Dr. Rife: Real Time PCR. To confirm the rtPCR products observed where actually RBCL an 2% agarose gel was ran to measure the size of the product. Protocol for the gel was from Dr. Rife: Agarose Gel Electrophoresis of DNA.