Canine Brucella

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Basic Kit
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Simplex Primer Kit for PCR 100* $225.00 $650.00
Duplex Primer Kit for PCR 100* $260.00 $750.00
Multiplex Primer Kit for PCR 100* $350.00 $1,500.00
Primer Kit for RT-PCR 100* $225.00 $1,500.00

ELISA for lab or field - Troubleshooting
(Enzyme Linked Immunosorbant Assay)
 
High Background

Possible cause: Insufficient washing, improperly made wash solution, or old wash solution.

Solution: During each washing step, be sure that each well receives wash solution. When adding the solution, it should be allowed to gently swirl and fill each well. Each well should be washed at least three times between steps. As a rule, the plate can never be washed too much only too hard. After the last wash has been completed, any solution remaining in the plate should be further removed by banging the plate against a cloth or paper towel. The wash solution must be diluted as per instructions in the protocol. Be sure to add the proper amount of Tween to each batch of wash solution. It is best to make up the wash solution the day it will be used. If this is not possible, the solution should be no older than 7 days.

Possible cause: Substrate is not fresh.

Solution: Add the hydrogen peroxide to the ABTS solution just before it is to be added to the wells. After the addition of hydrogen peroxide, the solution must be used within 30 minutes.

Possible cause: Partial contamination of the substrate with the conjugate.

Solution: Change pipette tips between the additions of each reagent. If using reservoir trays, separate trays should be designated for each reagent. Carry over from using the same tray for each reagent can contribute to contamination. If the bottle of ABTS becomes contaminated it will turn a dark green similar to the color of the high positive control or darker. Be aware that the solution does have a green tint to it. This is ok for use.

(To test whether the ABTS is still good, using a clean test tube, add 100 to 200µl of ABTS and 1µl of hydrogen peroxide. Shake well then add 10µl of Streptavidin HRP. Color development should occur within a few minutes.)

 

Rogue wells/ Variation between duplicates:

Possible cause: Improper washing

Solution: During each washing step, be sure that each well receives wash solution. When adding the solution, it should be allowed to gently swirl and fill each well. Each well should be washed at least three times between steps. As a rule, the plate can never be washed too much only too hard. After the last wash has been completed, any solution remaining in the plate should be further removed by banging the plate against a cloth or paper towel. The wash solution must be diluted as per instructions in the protocol. Be sure to add the proper amount of Tween to each batch of wash solution. It is best to make up the wash solution the day it will be used. If this is not possible, the solution should be no older than 7 days.

Possible cause: Pipetting errors.

Solution: Each well should receive the same amount of sample/reagent. Any variation in volumes from well to well can lead to variations in signal intensity at the end. See section titled "Tips" for more information on proper pipetting techniques and the proper way to dispense solutions from a dropper bottle.

 

All wells including negative controls turned green/blue

Possible cause: Contamination of the substrate with the conjugate.

Solution: Change pipette tips between the additions of each reagent. If using reservoir trays, separate trays should be designated for each reagent. Carry over from using the same tray for each reagent can contribute to contamination. If the bottle of ABTS becomes contaminated it will turn a dark green similar to the color of the high positive control or darker). Be aware that the solution does have a green tint to it. This is ok for use.

Possible cause: Contamination between samples or controls.

Solution: Always change pipette tips in between samples, controls and all other reagents. It is best to work in order starting from known negatives, to unknown samples and ending with known or high suspect positive samples. When preparing samples for testing, change gloves between samples. If this is not possible, wash hands or gloves thoroughly between samples.

Possible cause: Substrate is not fresh.

Solution: Add the hydrogen peroxide to the ABTS solution just before it is to be added to the wells. After the addition of hydrogen peroxide the solution must be used with in 30 minutes.

No Color. Even controls did not produce a signal

Possible cause: Reagents were forgotten or added in the wrong order.

Solution: Keep track of what steps have been completed. Do not leave out any step or switch the order.

Possible cause: Protocol times were not followed.

Solution: Be sure to pay close attention to times set forth in each protocol. Deviations from the protocol can cause false positive or false negative results.

Possible cause: : If using ABTS, hydrogen peroxide may not have been added.

The hydrogen peroxide is necessary in order for the substrate and the conjugate to react. Be sure this is added to the ABTS before use.

Unexpected low readings

Possible cause: Washing was done too hard.

Solution: The wash solution should be allowed to swirl gently into each well. The stream of wash solution should be hard enough to wash away any bubbles or debris that may be in the wells but not so hard that it will strip away the antibody coated onto the plate or disrupt any specifically bound reagents.

Possible cause: Reagents not warmed to room temperature before use.

 

Solution: All reagents must be at room temperature before use. Remove the solutions from the refrigerator ahead of time and place in an area away from direct sunlight. Each solution should be warm to the touch before use.

Possible cause: Wrong filter used to take readings.

Solution: Check to see if the proper filter is being used. Each substrate will have a different absorbance reading. Refer to the insert for correct filter information.

Unexpected high readings

Possible cause: Insufficient washing.

Solution: During each washing step, be sure that each well receives wash solution. When adding the solution, it should be allowed to swirl and fill each well. Each well should be washed at least three times in between steps. The wash solution must be diluted as per instructions in the protocol. Be sure to add Tween to each batch. It is best to make up the wash solution the day it will be used. If this is not possible, the solution should be no older than 7 days.

Possible cause: Wrong filter used to take readings.

Solution: : Check to see if the proper filter is being used. Each substrate will have a different absorbance reading. Refer to the insert for correct filter information.


Go to Tips
 

Top
Dot Blot - Troubleshooting
High Background

Possible cause: Each solution was not completely removed before subsequent reagents were added.

Solution: Remove as much of the solution as possible before addition of the next reagent. This is especially important after the addition of the Anti-Dig/AP conjugate. The development solution reacts with the AP in the conjugate. The AP will react whether it is specifically bound to the DNA probe or remaining in the bag due to insufficient washing. Washing steps are included in the protocol to assist in removing non-reacted reagents.

Possible cause: Membranes were allowed to dry out at some point during testing.

Solution: Be sure that each solution covers the entire membrane for the duration of each incubation step. The membrane should not be allowed to dry out between steps. To ensure that the membrane will remain covered by the solutions, after removing bubbles, fold the bag down until it is just above the edge of the membrane.

Light circles or fuzzy areas on the membrane

Possible cause: Light circles can be caused by bubbles in the solution remaining in the bag during incubation periods.

Solution: Remove all bubbles from the bag before beginning incubations. If all bubbles cannot be removed, be sure that they do not sit on top of the membrane. This can be done by laying the bag flat and gently pushing the bubbles towards the top of the bag and away from the membrane.

Possible cause: Mishandling of the membrane either by touching the membrane with bare hands, using gloves containing powder, or folding the membrane

Solution: Never touch the membrane with bare hands. Always use powder-free gloves. Keep the protective paper on the membrane until use. When handling the membrane, it is best to hold it by the edges only using gloved hands or clean forceps.

Possible cause: Membranes were allowed to overlap during testing.

Solution: Do not allow the membranes to overlap during any incubation step. This is especially important (with the membranes in the bag that receives hybridization solution) during the hybridization and development steps. Overlapping of the membranes will prohibit solutions from reaching all sections of each membrane.

No positive signal seen even with control membranes

Possible cause: Reagents not warmed to room temperature before use (unless otherwise stated in the protocol).

Solution: Remove reagents from refrigerator and allow them to warm to room temperature completely before they are used. Any reagents that require further heating need to be at their required temperature before use.

Possible cause: Insufficient denaturation of the probe.

Solution: The probe must be placed directly in boiling water for 10 minutes. A reduction in time or temperature may inhibit the two strands of the probe from completely separating.

Possible cause: Insufficient cooling of the probe.

Solution: After boiling the probe it must be immediately placed at 4°C for 10 minutes before it can be added to the membrane. This drastic cooling process keeps the two separated strands of the probe from coming back together until they can be added to the membrane.

Possible cause: Incorrect hybridization temperature or time.

Solution: The incubation temperature specified in each protocol must be maintained for the duration of the hybridization step. Place a thermometer next to the bags to ensure that the temperature in the area where the bags are incubated is at the proper temperature. Depending on the size of the incubation oven used, temperatures can vary inside the unit from shelf to shelf. Adjust the temperature as needed. Also be sure to maintain the correct temperature for the entire three hours as stated in the protocol.

Possible cause: One or more of the reagents were not added or used in the incorrect order.

Solution: Keep track of what steps have been completed. Do not leave out any step or switch the order.

Possible cause: Protocol times and temperature were not followed.

Solution: Be sure to pay close attention to times and temperatures set forth in each protocol. Deviations from the protocol can cause false positive or false negative results.


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Top
In Situ/Immunohistochemistry - Troubleshooting
Poor tissue quality
Problem: Tissue sections fall off slide during testing.

Solution: Positively charged slides must be used when preparing slides (see below). During the Proteinase K step, do not allow the incubation period to exceed the time noted in the protocol. Allowing the Proteinase K to stay on the slides too long can cause the tissue to be degraded and fall off. The tissue is very vulnerable during and just after this step so care should be taken when adding and removing reagents and/or cover slips.

Problem: Tissue sections are badly disrupted at the end of the test.

Solution: The entire in situ procedure is hard on the tissue. Proteins that hold the sections together are being weakened. Special care and consideration should be taken thorough out the procedure. See the section titled " Tips" for more information on how to properly add reagents, wash slides and remove cover slips with limited distress to the tissue.

Too much background or nonspecific staining
Possible cause: Tissue was badly disrupted leading to many areas were the development solution can be trapped.

Solution: Care should be taken throughout the process to avoid excess tissue damage.

Note: Nonspecific staining can be easily distinguished by it having no cellular morphology. Cuticular staining and areas where the reagents were nonspecifically trapped during testing, will usually have a uniform appearance and will not show any cell association or morphology.


Possible cause: Tissue sections were allowed to dry out at some point during testing.

Solution: Once the test has begun, the tissue section should never be allowed to dry out. Each solution must cover, and remain covering the entire tissue section during the entire incubation period.

Possible cause: Development solution remained on the slides in excess of two hours or development was carried out in the light.

Solution: The development solution should be removed and inactivated after two hours. During incubation, the slides should be placed in a humid chamber and covered or placed in a dark area away from all light.

No staining seen even on positive control slides
Possible cause: Reagents not warmed to room temperature before use. (unless otherwise stated in the protocol).

Solution: Remove reagents from refrigerator and allow them to warm to room temperature completely before they are used. Any reagents that require further heating need to be at their required temperature before use.

Possible cause: Insufficient denaturation of the probe.

Solution: The probe must be placed directly in boiling water for 10 minutes. A reduction in time or temperature may inhibit the two strands of the probe from completely separating.

Possible cause: Insufficient cooling of the probe.

Solution: After boiling the probe it must be immediately placed at 4°C for 10 minutes before it can be added to the slides. This drastic cooling process keeps the two separated strands of the probe from coming back together until they can be added to the slides.

Possible cause: Incorrect hybridization temperature or time.

Solution: The incubation temperature specified in each protocol must be maintained for the duration of the hybridization step. Place a thermometer next to the slides to ensure that the temperature in the area where the slides are incubated is at the proper temperature. Depending on the size of the incubation oven used, temperatures can vary inside the unit from shelf to shelf. Adjust the temperature as needed. Also be sure to maintain the correct temperature for the duration of hybridization as stated in the protocol.

Possible cause: One or more of the reagents were not added or used in the incorrect order

Solution: Keep track of what steps have been completed. Do not leave out any step or switch the order.

Possible cause: Protocol times and temperature were not followed.

Solution: Be sure to pay close attention to times and temperatures set forth in each protocol. Deviations from the protocol can cause false positive or false negative results.


Possible cause: Insufficient removal of the wax during rehydration

Solution: Each slide should be viewed after the 65°C melting step to be sure that the wax appears melted. As quickly as possible the slides should be taken from the incubator and placed directly into xylene or xylene substitute. If the xylene or xylene substitute is being reused, and there is a problem getting all of the wax off of the slides, the xylene should be discarded and replaced with fresh solution.

Possible cause: Proteinase K is not active.

Solution: Repeated freezing and thawing of the Proteinase K solution will result in loss of activity. If the entire solution is not being used, the remaining solution should be divided into smaller volumes and frozen. When ready to use, remove only the amount needed from the freezer and thaw on ice. Keep the solution on ice until it is ready to be added to the slides. Proteinase K is used to weaken tissues and cell walls, allowing the reagents access to target DNA.