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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 = A/A
Heterozygote = G/A
Wild type = G/G
GCAGTTTTACAGCGCAAAGAGGAGGAGGATCGCCAGATG
AAGCAACTTGTCCAAGCCTTA(C/T)AAGTCTCACTAGAGAAAG
AAAAGATGGAAGTGAACAGCCTGAAGGAACAGg
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 18930 | Fluorophore | GAT CGC CAG ATG AAG CAA CTT G | Forward | |||
| 18931 | TTC AGG CTG TTC ACT TCC ATC TT | Reverse | ||||
| 18932 | TTT TCT TTC TCT AGT GAG AC | SEQ |
| Component | Final Concentration |
|---|---|
| ddH2O | |
| Kapa 2G HS buffer | 1.00 |
| MgCl2 | 2.00 |
| dNTPS-kapa | 0.20 |
| Primer 1 | 0.50 |
| Primer 2 | 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 | -- | -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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