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This assay will NOT distinguish hemizygous from homozygous trangenic animals. This assay only confirms the point mutation.
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 |
|---|---|---|---|---|---|---|
| 13068 | GGC TAC CAA AGG TGC AGA TAA | Forward | ||||
| 13069 | Fluorophore | ATG CAG TGT CTC GCA AGT GTA | Reverse | |||
| 13070 | CGG CAG TGT GAG TAC CT | 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 |
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