For in-depth product & services help, ask our
Technical Information Scientists
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 |
|---|---|---|---|---|---|---|
| 15675 | CTC TAC TGG TTC CCG CTC AA | Forward | ||||
| 15676 | Fluorophore | ATG ACC ATC AGG AGC AAC AGC | Reverse | |||
| 15677 | GCT CAA GGT TCT GTA CG | 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 |