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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.
Mutant = T
Heterozygote = T/A
Wild type = A
X-linked mutation
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 11552 | CAC AGT GGA TTG ATT TCG GTA TT | |||||
| 11553 | Fluorophore | CCA TTG GAA GAC GCT GAA TTA | ||||
| 11554 | TGC CAA GAT CCA CTT T |
| Component | Final Concentration |
|---|---|
| ddH2O | |
| Kapa 2G HS buffer | 1.00 |
| MgCl2 | 2.00 |
| dNTPS-kapa | 0.20 |
| 11552 | 0.50 |
| 11553 | 0.50 |
| Glycerol | 5.00 |
| Kapa 2G HS taq polym | 0.01 |
| DNA |
| Step | Temp °C | Time | Note |
|---|---|---|---|
| 1 | 94.0 | -- | |
| 2 | 94.0 | -- | |
| 3 | 65.0 | -- | -0.5 C per cycle decrease |
| 4 | 68.0 | -- | |
| 5 | -- | repeat steps 2-4 for 10 cycles | |
| 6 | 94.0 | -- | |
| 7 | 60.0 | -- | |
| 8 | 72.0 | -- | |
| 9 | -- | repeat steps 6-8 for 28 cycles | |
| 10 | 72.0 | -- | |
| 11 | 10.0 | -- | hold |