DON Protocols
Michigan State Analyses (PNUS, NUS, SUS) Sample Prep:
1. Weigh out 5g of seed per sample.
2. Place each sample in a plastic bag and label with:
a. Dave Van Sanford
b. University of Kentucky
c. PNUS, NUS, or SUS
d. Entry # (1000 + Entry # = Rep1, 2000 + Entry # = Rep2)
e. If needed, designate P for Princeton and L for Lexington.
3. Grind sample with coffee grinder 10 seconds.
WEAR GLOVES AND DUST MASK!!
4. Place flour back into the plastic bag.
5. Vacuum out the grinder and make sure the blade and lid are
clean for each sample.
6. Mail to:
Ms. Carmen Medina
Dept. Plant Pathology
107 CIPS Building
Michigan State University
East Lansing, MI 48824-1311
In House Analyses (WYT) Sample Prep:
1. Order DON kit from Diagnostix 1-800-282-4075, rep is Bob
Reynolds.
2. Take small sample from Rep1 and Rep2 for each location,
separately (LEX and PRL).
3. Combine reps so there is one sample for each location.
4. Weigh out 5g per sample, put in a plastic bag and label it.
5. Grind sample with coffee grinder 10 seconds.
WEAR GLOVES AND DUST MASK!!
6. Place flour in a labeled 50 mL centrifuge tube.
7. Clean the grinder so it’s clean for the next sample.
8. Samples can be stored up to 1 week in the fridge.
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Diagnostix EZ-Quant Vomitoxin (DON) Plate Kit Protocol
Day prior or morning before running test:
1. Put flour into labeled 50 mL tubes.
2. Label vials and fit with filter paper folded into cones.
Day of:
1. Take reagents out of the refrigerator so they can get to room
temperature before use.
2. Add 25 mL dH20 to the flour in 50 mL centrifuge tubes.
3. Place tubes on the shaker for 10 min at 200 rpm.
4. Make Wash Solution by adding concentrate to dH20 for a final
volume of 1L and mix.
5. Fill 2 wash bottles with wash solution (1L total volume).
6. Find 2 trays for DON test wells.
7. Allow the solution to settle a moment after it was shaken.
8. Pour some of the solution into the corresponding filter paper
cones so that there is 100 µl of solution for the ELISA.
Running the Test
1. Dispense 100L calibrators and samples into red mixing wells. Put
the calibrators in the first 5 (A1-5) and last 5 (H8-12) wells of the
plate in order (0 to 6 ppm).
2. Using multi-channel pipette, dispense 100L of Enzyme Conjugate
into red mixing wells and create a homogenous mixture by
repeatedly pipetting up and down 5X.
3. Using multi-channel pipette, transfer 100L of the reaction mixture
into clear test wells. Discard the red mixing wells into an
appropriate waste container.
4. Incubate the clear test wells for 10 minutes.
5. Dump the contents of the wells into a waste container and carefully
shake out any residual solution. Using wash bottle, fill the wells to
overflowing with Wash Solution, dump wash and carefully shake out
any residual solution. Repeat 4X for a total of 5 washes. Following
the last wash, tap the inverted wells onto absorbent paper to
remove the last of the wash solution.
6. After the final wash, tap the strips repeatedly onto paper towel to
remove excess wash. After tapping, check for large bubbles, which
should be burst with a clean pipette tip and tapped out again (use
toothpicks).
7. Using the multi-channel pipette, dispense 100L of Substrate into
each well.
8. Incubate the wells for 5 minutes.
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9. Using the multi-channel pipette, dispense 100ul of Stop Solution/
well.
10. Within 10 minutes read and record absorbency of each well at
450 nm. To be valid, the 0 ppm calibrator should have absorbency
value between 0.6 and 2.5.
11. Input values in the DON ELISA Workbook Excel file. The R2
value should be between -0.9920 and -1.0000.
ELISA Plate Loading
1. Use worksheet to write down order (ELISA worksheet under C
drive, Scab Data, Don)
2. Add appropriate standards (as shown on worksheet)
3. Add samples and follow color coded DON worksheet
Reading ELISA plate
1. Put plate into reader with A1 in top left corner
2. Follow instructions on computer
3. Print out absorbance
4. Enter into Excel worksheet provided by manufacturer (DON
ELISA workbook) to get DON ppm
Place anything with flour or DON into 10% bleach and throw away.
DO NOT put HCl into bleach solution.
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