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Matrix Specific Protocol Roundup Ready ® (CP4 EPSPS) ImmunoStrip® Catalog number: 74000 Alfalfa leaves and seed testing YOU WILL NEED INTENDED USE Sample extract buffer SEB4 (ACC 00958 or ACC 01958) Micropipettes Sterile micropipette tips Graduated cylinder Balance 1-500 grams Distilled water Conical micro tubes or microcentrifuge tubes Micro tube rack Sarstedt polypropylene 13 ml tubes Chrome grade 25, ½ inch metal balls Tube rack Sample mesh bags (ACC 00930) Small letter holder This kit is intended for seed quality purposes to determine the presence of the CP4 EPSPS protein in leaves and seeds of alfalfa. The expression of CP4 EPSPS ® ® transgenic protein in plants results in Roundup herbicide resistance. Roundup is a broad spectrum herbicide used to control weeds. Using this test system, you can reliably test individual alfalfa leaves or detect 1 transgenic CP4 EPSPS seed in 100 seeds (1%) of alfalfa. ® Roundup Ready is a registered trademark of Monsanto Company. SAMPLE PREPARATION Leaves, seedlings, or seeds must be ground well and diluted in SEB4 sample extract buffer. Use the following sample preparation guidelines for specific tissue types. Individual leaf samples A simple method for grinding a single leaf sample is by using Agdia’s SEB4 buffer filled sample extraction bags (ACC 00958). Use only one sample per bag and be sure to label each bag. Each filled bag contains 3 ml of sample buffer. The recommended 1:20 dilution would require about 0.15 g leaf sample. Place the sample between the mesh linings of the bag. Rub the bag with a pen to completely crush the sample and mix the contents uniformly. Composite seed samples The provided grinding guidelines have been optimized for manually shaking in tubes (13 ml) with two metal balls (1/2 inch Chrome Grade 25). If using SEB4 dry buffer powder, prepare enough 1X buffer for the number of composite samples you will be performing. Determine the average weight of your seed sample. A composite sample of 100 seeds will weigh about 0.25 g. Put each sample in a dry polypropylene tube and add 2 metal balls to each tube, being sure that the cap is completely closed. Label the tubes as needed. Hold the tube as shown, turning the tube upside down so that the cap is facing downward. Shake each tube with three up and down movements per second for a total of 2 minutes. This process should adequately crush the seeds. Visually check the tube contents for any unground seed. Remove the cap from the tube and add the buffer at the recommended 1:20 dilution. Close lid and gently mix the sample with the buffer by inversion for 5 - 10 seconds. Do not shake vigorously. msp001 Matrix Specific Protocol Roundup Ready ® (CP4 EPSPS) ImmunoStrip® Catalog number: 74000 Alfalfa leaves and seed testing TEST PROCEDURE Let the seed or leaf extract sit for at least 5 minutes before testing with the ImmunoStrip. Use a small letter holder for the sample bags or use a tube rack for composite sampling. Transfer 500 µl of the supernatant (liquid phase) to a conical microtube. When handling the strips, always grasp the top of the strip marked with the test name. Do not remove protective covering. Keeping the strips in a vertical position, insert the ends of the strips marked “sample” into the extract of the microtube or bag. Do not allow much more than 0.5 cm or ¼ inch of the ends of the strips to be submerged in the extract. Be sure the strips remain in the extract during the test The control line will appear in 3 to 5 minutes. Maximum reaction occurs in 20 minutes at which time the ImmunoStrip should be removed from the buffer. The control line assures that the test is working properly. If the control line does not appear, the test is invalid. Refer to the product insert for interpreting results. LIMITATIONS The following limitation is in addition to those stated in the product insert: msp001 Seed Grinding: All seeds must be ground to a fine powder. Seed grinding guidelines described in this technical note are optimized for manually shaking of the tubes with 2 metal balls. If using a mechanical shaker, the strength of the tubes and the number of metal balls must be considered. Tubes can easily break. Other labs using such mechanical methods recommend using only one metal ball per tube. Power and time play a key role in successful seed grinding.