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.
Ctgggaatatggctgcctaagggagagggcctccctggaaagatgtgtgcagtatgtctcgtatgcagaaatacccactctgaccaggttgcccggggcctaggtctcccatcctttccttccggttgctctggagatcctaagagccctgaagctaaggcaaccacgtgcctgtcatctccctcagggagtacagtctgcctcctgtcttcaggacctgctataatgacctcttcatcttctctttgctccctagaggccattaactgtctgat[G/A]agagcaattgagatctatacagacatggtaagggttgcttgctgatcctgccttgctctgtcctctcctgtttatgctgtgctttagagggcagggatttgatcgctgagctgagctacagaacggggtgtgtgctgcagcctctgctcatgtcctcatgcacatgctgcctctgtccccttcgtgatccccttgtggctgcagtgtccatgcagtacttcccttgcctggccctcccctgatgtccctaggagcctgagaaatgctcagtctccccctctaggaaggctactatcctgagagacagc
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
13555 | Fluorophore | CTG TCT TCA GGA CCT GCT ATA ATG | ||||
13556 | AGC TCA GCG ATC AAA TCC C | |||||
13557 | CTG TAT AGA TCT CAA TTG CT |
Component | Final Concentration |
---|---|
ddH2O | |
Kapa 2G HS buffer | 1.00 |
MgCl2 | 2.00 |
dNTPS-kapa | 0.20 |
13555 | 0.50 |
13556 | 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 |