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| Real-time PCR |
- 1.When Internal Control cannot be seen
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First of all, please check whether correct fluorophore was selected appropriately for data analysis and check whether the test proceeded under the guideline of the protocol. If all is correctly set, then the problem may be related to poor PCR reaction or an extraction problem caused by the PCR inhibitor. Proceed with PCR again after diluting the template by 10 to 100 times in distilled water (for PCR inhibitor problem) or re-check using the same sample with another aliquot or carry out the test again entirely after re-extraction.
- 2.When signals are observed in the Negative Control or
when false result is observed
- There could be cross contamination during extraction or PCR process. Cleanse the work area and equipments using sodium hypochlorite and ethanol and perform the whole process again from extraction of nucleic acid.
- 3.When signals cannot be observed at the Positive Control or
when false results are observed
- First of all, please check whether correct fluorophore was selected appropriately for data analysis and check whether the test proceeded under the guideline of the protocol along with the use by date of the drug
- 4.When Internal Control signal is not observed during RT-PCR test
- First of all, please check whether correct fluorophore was selected appropriately for data analysis. If all is correctly set, then the problem may be related to poor PCR reaction or the extracted RNA amount is not adequate. Proceed with RT_PCR again after diluting the template’s RNA by 2 to 5 times in DEPC-dH2O (for PCR inhibitor problem) or re-check using the same sample with another aliquot or carry out the test again entirely after re-extracting the RNA of higher purity.
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| Multiplex PCR |
- 1. When Internal Control band cannot be seen during the RT-PCR test
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First of all, check the quantity of RNA. RNA with lower purity can cause poor performance of RT_PCR. Proceed with RT_PCR again after diluting the template’s RNA by 2 to 5 times in DEPC-dH2O (for PCR inhibitor problem) or re-check using the same sample with another aliquot or carry out the test again entirely after re-extracting the adequate amount of RNA.
- 2. When Internal Control band cannot be observed
- Check whether test was performed according to the guideline. If all is well, there is a possibility of PCR inhibitor causing the problem. Proceed with PCR again after diluting the template by 10 to 100 times in distilled water
- 3. When PCR band cannot be observed on the agarose gel
- Band cannot be checked in cases where EtBr dye is not well or when the band is seen to be excessively exposed to UV. In such a case, perform 1/10,000 dilution for 30 minutes in the shaker with 1100 ± 100 rpm and shake in the EtBr of 10mg/ml.
PCR band may not appear also when the quantity of pathogen is excessive. Perform the PCR again after diluting by 2 to 5 times.
- 4. When signals are observed at the Negative Control or false result is observed
- There could be cross contamination during extraction or PCR process. Cleanse the work area and equipments using sodium hypochlorite and ethanol and perform the whole process again from extraction of nucleic acid.
- 5. When signals are not observed at the Positive Control or
false results are observed
- First of all, please check whether correct fluorophore was selected appropriately for data analysis and check whether the test proceeded under the guideline of the protocol along with the use by date of the drug.
- 6. What is the required level of maintenance?
- Clean the inside with tissues after spraying the inside with 70% of ethanol. After cleansing with ethanol, keep the UV lamp turned on.
- 7. The UV lamp of the equipment should be replaced periodically.
What is the replacement cost and what is the life span of the lamp?
- Usage period may be shorter where the lamp is turned and turned off frequently but the average life span of the lamp is approximately 8000 hours. When turned on for 24 hours non-stop, it can be used for about 1 year. The lamp cost around 30,000 won each. The power required for the lamp is 20W.
- 8. What is the required level of maintenance?
- Clean the inside with tissues after spraying the inside with 70% of ethanol. After cleansing with ethanol, keep the UV lamp turned on. For other maintenance activities, please check the maintenance table provided by Seegene.
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