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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.
For unknown reason, this protocol cannot distinguish het from hom and can only be used to confirm the point mutation.
Sequencing primer anneals to the forward strand.
Only red mutation is highlighted in pyrosequencing data. The blue mutation is not highlighted but can be read from the result. KC note 1-5-2012.
underlined amino acid code: WT = GAG; E64D = GAC; E64G = GGC
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
13097 | ATA GGG GTG CTG ACA TCG A | Forward | ||||
13098 | Fluorophore | GAA GGT TCC CAG CTG TTC AG | Reverse | |||
13099 | GAT ACC ATT TAT GAT GAA GA | SEQ |
Component | Final Concentration |
---|---|
ddH2O | |
Kapa 2G HS buffer | 1.00 |
MgCl2 | 2.00 |
dNTPS-kapa | 0.20 |
13097 | 0.50 |
13098 | 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 |