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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.
CCTGAAGCCCCACAATGTGCTGCTTTTTACCCTGTA
TCCCAATGCTGCCATCATTGCGAAGATT[g/a]CGGAC
TACGGGATCGCACAGTACTGCTGCAGGATGGGAATA
AAGACATCAGAGGGCACCCCAGGTAGGAGAACAAGT
TTACTTTTTTTGCAGTGGACCATCAGTGCCCTCAAG
TGTGGGTC
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
17644 | GAA GCC CCA CAA TGT GCT G | |||||
17645 | Fluorophore | TAC TGT GCG ATC CCG TAG TCC | ||||
17646 | CCA TCA TTG CGA AGA TT |
Component | Final Concentration |
---|---|
ddH2O | |
Kapa 2G HS buffer | 1.00 |
MgCl2 | 2.00 |
dNTPS-kapa | 0.20 |
17644 | 0.50 |
17645 | 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 |