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.
Gtaaaatagagctagtgcttcagcacatctccgttacttttagaagctggttttcactaatgttcttctgttctcag
GCTTGTCATTGGAAGAGCAGTTGCGGAGATTACAAGAAGAACGAACTTGCaaagtgtg
tatggacagagaggtttctattgtgttcattccgtgtggt(C/g)(A/c)(T/a)CTAGTAGTCTGCCAGGA
ATGTGCCCCTTCTCTAAGGAAGTGCCCCATCTGCAGGGGGACAATCAAGGGGACTGT
GCGCACATTTCTCTCATGAGTGAAGAATGGTCTGAAAGTATTGTTGGACATCAGAAG
CTGTCAGAACAAAGAATGAACTACTGATTTCAGCTCTTCAGCAGGACATTCTACTCTC
TTTCAAGATTAGTAATCTTGCTTTATGAAGGGT
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
17615 | TCT ATT GTG TTC ATT CCG TGT GG | Forward | ||||
17616 | Fluorophore | TGA TTG TCC CCC TGC AGA T | Reverse | |||
17617 | TTC ATT CCG TGT GGT | SEQ |
Component | Final Concentration |
---|---|
ddH2O | |
Kapa 2G HS buffer | 1.00 |
MgCl2 | 2.00 |
dNTPS-kapa | 0.20 |
17615 | 0.50 |
17616 | 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 |