| Proublems |
Possible Cause(s) |
Recommendation(s) |
| a. |
No amplification can be monitored |
Wrong channel has been chosen. |
If the quantitative PCR instrument you use which automatically detects fluorescence intensities of all the channels, you can simply reset the right plate information and channel (FAM/VIC) to recalculate data.
If the instrument does not automatically collect whole channels, you need to abort and redo the run. |
| Pipetting errors or omitted reagents. |
- Check the set-up of the reaction and missing reagents.
- Redo the PCR run.
- Always run a positive control along with samples.
|
| Wrong program. |
- Check the amplification program which is followed as the package insert.
|
| Instrument broken down. |
- Contact the technical support of instrument’s company.
|
| b. |
Weaker fluorescent signal |
Using Heparin as the anticoagulant in blood collection. |
- Use either sodium citrate or EDTA as the anticoagulants in blood collection.
- Extract DNA within 3 days after blood withdrawing.
|
| Inhibitory effects on PCR reaction. |
- Decrease the gDNA loading amount.
- Qiagen QIAamp® DNA Blood Mini Kit is recommended for DNA extraction.
|
| With Inappropriate gDNA amounts. |
- A total gDNA of 25~100 ng/reaction is appropriate for these Detection Kits.
|
| Poor gDNA quality |
- The OD260/280 ratio between 1.7 and 2.0 is required for performing these Detection Kits.
- Clean up samples by a purification kit or re-extract DNA from blood.
- Qiagen QIAamp® DNA Blood Mini Kit is recommended for DNA extraction.
|
| Very low starting amount of DNA |
- Qiagen QIAamp® DNA Blood Mini Kit is recommended for DNA extraction.
- A total gDNA of 25~100 ng/reaction is appropriate for these Detection Kits.
|
| Freeze and thaw reagents more than three cycles |
|
| Reagents are kept in inappropriate temperature |
- Keep kits in -20°C for long-term storage.
- Keep the reagents on ice when thaw them.
|
| Processing reaction under direct lighting |
- Prepare the reagent as soon as possible.
- Keep PCR Master Mix away from light.
|
| c. |
Higher signal shown in NTC |
Contaminations |
- Repeat the run.
- Always wear gloves when preparing the reactions.
|
| d. |
Sample’s signal is much lower than positive controls |
Components are not homogeneously mixed |
- Thaw and vortex components completely .
- Vortex the vial for 15 seconds to mix the PCR Master Mix thoroughly.
|
| Pipetting errors or omitted reagents |
- Check for missing reagents and reset the reaction.
|
| e. |
Fluorescent signal varies |
Pipetting errors or omitted reagents |
- Check for missing reagents and reset the reaction.
|
| Poor gDNA quality |
- The OD260/280 ratio between 1.7 and 2.0 is required for performing these Detection Kits.
- Clean up samples by a purification kit or re-extract DNA from blood.
- Qiagen QIAamp® DNA Blood Mini Kit is recommended for DNA extraction.
|
| Very low starting amount of DNA |
- Qiagen QIAamp® DNA Blood Mini Kit is recommended for DNA extraction.
- A total gDNA of 25~100 ng/reaction is appropriate for these Detection Kits.
|
| Components are not homogeneously mixed |
- Mix components completely.
|
| Contaminations |
- Repeat the run.
- Always wear gloves when preparing the reactions.
|
| Reaction solution is not in the bottom of tube. |
- Leave reaction solution in the bottom of tube by higher centrifugal speed.
|
| Instrument broken down |
- Contact the technical support of instrument’s company.
|
