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This genotyping assay uses pyrosequencing technology and is run on the Biotage PSQ 96MA. The Jackson Laboratory is not posting the complete details of our pyrosequencing genotyping assays as the primers for pyrosequencing cannot be used for sequencing using more traditional methods. The wild type and mutant nucleotides and the flanking DNA sequence are provided below.
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 12558 | Fluorophore | CCG CAC ACA CCA GCA TAT TC | Forward | |||
| 12559 | GGT GGG TGT TGA TAG AGG CAT AG | Reverse | ||||
| 12560 | GGT GTT GAT AGA GGC ATA | SEQ |
| Component | Final Concentration |
|---|---|
| ddH2O | |
| Kapa 2G HS buffer | 1.00 |
| MgCl2 | 2.00 |
| dNTPS-kapa | 0.20 |
| Primer 1 | 0.25 |
| Primer 2 | 0.25 |
| Kapa 2G HS taq polym | 0.01 |
| DNA | 0.00 |
| Step | Temp °C | Time | Note |
|---|---|---|---|
| 1 | 94.0 | -- | |
| 2 | 94.0 | -- | |
| 3 | 65.0 | -- | -1.5 C per cycle decrease |
| 4 | 68.0 | -- | |
| 5 | -- | Go to step 2-4, 10 times | |
| 6 | 94.0 | -- | |
| 7 | 50.0 | -- | |
| 8 | 72.0 | -- | |
| 9 | -- | Go to step 6-8, 28 times | |
| 10 | 72.0 | -- | |
| 11 | 10.0 | -- | hold |