This article provides an overview of the most common issues with Melt Assay experiments, including causes and solutions. Contact us if you need additional assistance.
General Melt Assay troubleshooting
The table below applies to all Melt Assays.
| Problem | Cause(s) | Solution(s) |
|---|---|---|
| No specific melt curves visible in any reaction. * | Programming error(s) in thermocycler/melt program. | Correct errors in the program and repeat the Melt Assay. |
| No polymerase enzyme included in the master mix. | Repeat the Melt Assay. | |
| No specific melt curve visible in some reactions. | PCR inhibitors present in the DNA samples. | Repeat the Melt Assay using 5-fold diluted sample, or with a newly prepared sample. |
| Q-fragment peak is the highest melt peak. * | Insufficient sample DNA. | Repeat the Melt Assay using a more concentrated sample. |
| No or only a very low Q-fragment peak is present in the no-DNA reaction. | PCR reaction failed, possibly due to PCR inhibitors present in the no-DNA reaction. | Check reaction content and repeat the Melt Assay. |
| In one or more samples, the observed Tm of the target peaks is slightly lower or higher than expected. | Evaporation of reactions results in an increased salt and glycerol concentration. This can result in a slightly altered Tm. | Repeat the Melt Assay when the difference in Tm with similar samples is more than 1°C for all the target peaks. |
| Melt peaks of all reactions are low. | Wrong type of plate/strips/seal used. Probe signals are higher with correct plate/seals as compared to strips with caps. | Use the recommended type of plates/seals. |
| Instrument failure. | Check instrument optics. | |
| The Q-fragment melt peak is not the only peak in the no-DNA reactions. | Contamination of reactions with amplicons from other samples or from previous experiments. |
Never open tubes, strips or plates after the PCR, especially not in the room where reactions are prepared. Do not discard used reaction tubes, strips and plates in the same room where the Melt Assay reactions are set up or where DNA samples are extracted. Use appropriate plates and seals and visually inspect the rims of the wells to ensure complete sealing. |
* For reactions with SALSA MC002 SMA Newborn Screen, also review the specific troubleshooting table below.
MC002-specific troubleshooting
The table below contains additional information that is specific to SALSA MC002 SMA Newborn Screen. The table with general information above also applies to reactions with MC002.
| Problem | Cause(s) | Solution(s) |
|---|---|---|
| No specific melt curves visible in any reaction. | NaOH concentration >50 mM. | Repeat the MC002 assay with the correct NaOH concentration. |
| Q-fragment peak is the highest melt peak. | Insufficient sample DNA present. | It is possible to reduce the volume of NaOH solution used for preparation of a crude extract, or to use a lower dilution factor for extracts from 3.2 mm punches. Alternatively, use extracts from washed punches (Protocol C in the instructions for use). Do not use more than 2 µl sample DNA per reaction as dilution of the reaction mixture will influence the Tm. |
| An extra melt peak is observed. | An extra melt peak with a Tm below 63°C can be the result of a SNP in either SMN1 or SMN2 where the probe binds. | No action needed in case a clear SMN1-specific melt peak at 63°C is present. When no clear SMN1 melt peak is visible, perform MLPA follow-up testing. |
| Melt peaks of reactions on crude extracts are much lower than the reactions on the SALSA SD074/SALSA SD075 samples. | Over-quenching of probe signals due to an oversaturated DBS. | When the Q-fragment specific melt peak is low or absent in most samples, the crude extracts can be further diluted by increasing the volume of NaOH solution used for preparation of a crude extract, or by using a higher dilution factor for extracts from 3.2 mm punches. Alternatively, use extracts from washed punches (Protocol C in the instructions for use). |
| Melt peaks of reactions on purified DNA are much lower than the reactions on SALSA SD074/SALSA SD075 samples. | DNA samples contain PCR inhibitors. | Test a different DNA purification method. |
| An SMN1-specific melt peak is clearly visible in the SALSA SD075 positive DNA sample reaction. | Contamination of reactions with amplicons from other samples or from previous experiments. |
Never open tubes, strips or plates after the PCR, especially not in the room where reactions are prepared. Do not discard used reaction tubes, strips and plates in the same room where the MC002 reactions are set up or where DNA samples are extracted. Use appropriate plates and seals and visually inspect the rims of the wells to ensure complete sealing. Be very careful with handling the sample plate in which the punch extracts are made. |