Download Matrix Specific Protocol

Matrix Specific Protocol
Roundup Ready ® (CP4 EPSPS) ImmunoStrip®
Catalog number: 74000
Alfalfa leaves and seed testing
YOU WILL NEED
INTENDED USE
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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
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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.
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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.
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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:
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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.