What if my library has indications of PCR-bubble formation?
What if my library has indications of PCR-bubble formation?
Sometimes, tissue with low RNA quality or very low cellular density can cause situations where the cDNA tapestation profile is normal, but the library trace shows evidence of very long PCR-bubble products on the trace (see below).
To fix this, you have 2 options:
1. Re-run the tagmentation reaction with fewer indexing PCR cycles - Run this with 8-9 cycles instead of the recommended 12 cycles.
IF THIS DOES NOT WORK, YOU CAN TRY OPTION 2 BELOW
2. Run a reconditioning PCR protocol using the library as template (see below):
| Component | 1 Reaction (μL) |
| Tagged library product | 2.5 |
| F-primer (from v2 kit, K006) | 2.5 |
| R-primer (from v2 kit, K006) | 2.5 |
| DNAse-free H2O | 10 |
| Nextera PCR Mix | 7.5 |
| Total | 25 |
3. Mix well by pipetting.
4. Briefly spin down the PCR tube.
5. Run the following PCR program:
| Temperature | Time | Cycles |
| 72ºC | 3 min | |
| 95ºC | 30 sec | |
| 95ºC | 10 sec | 3 Cycles |
| 55ºC | 30 sec | |
| 72ºC | 30 sec | |
| 72ºC | 5 min | |
| 4ºC | Hold |
6. Proceed with the same library cleanup and quantification steps in section "Library cleanup and quantification"