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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.
paste sequence here
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
19740 | AAT GTG TGC CAA CGA GAA TCA | Reverse | A | |||
19828 | Fluorophore-1 | GCC CAG TTT GAA AGC AAA CAC | Quencher-1 | Forward | A | |
19829 | Fluorophore-2 | CCA GGC TCG CGC TTC | Quencher-2 | WT Probe | ||
19830 | Fluorophore-3 | CCA GGC TGG CGC TTC | Quencher-3 | MUT Probe | A |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
19740 | 0.40 uM |
19828 | 0.40 uM |
Wt Probe | 0.15 uM |
Mutant Probe | 0.15 uM |
DNA |
Step | Temp °C | Time | Note |
---|---|---|---|
1 | 95.0 | -- | |
2 | 95.0 | -- | |
3 | 60.0 | -- | |
4 | -- | repeat steps 2-3 for 40 cycles | |
5 | 40.0 | -- | Forever |