Amplicon = 237 bp
HET = C/T
HOM (Ptprc<a>) = C/C
WT (Ptprc<b>) = T/T
TCAAACTATTATCTTAAGCAAGTAGCTGTGGATATAAACTATGTCTCATTAATTTTATTTTAATACTTACCACAGCAATACCTCAAGATACCAAAACACATGGCCAACTGTGGGAATATTCTGATAGAAGAGAATATTATTGGGTAGTCTTCAGATCAGTGGGATAACTAGCCAAAAAGAATCTATGAAAGAAATCTAATCTAAATGATCCAGGATAGGTCTAAGAACTTCACTGAGATTTTATTAATTCACAATTAATCAAAAGATAGGTATTCATGATTCAAGCTGGCTATGTAAATACTTCTTTTCAGTGTCTTATTTTTCCCATTATAACTTTTTCTGTTTTTAACAGTTCCAGAAACGCCTAAGCCTAGTTGTGGGGATCCAGCTGCAAGAAAAACGTTAGTCTCTTGGCCTGAGCCTG(T/C)ATCTAAACCTGA(G/T)(T/C)CTGCATCTAAACCCCATGGATATGTTTTATGCTATAAGAACAATTCAGGTAATGTAAAATTCCACTAGGGAAACAAAATCAAGAATTTTTAAATGTTGTAATTATTGTTTTAGCCAGGGAGCTACAAGGCTTTTATGAAGGTGATGAGAGATTCATGTTTGAGTTTGCTTAGATGAACACGCATCTCATGGAGAAGAATAAATATCTAGATCATTTATTAAAAATAGTGGACAGGACTAGAGTCCTTTAGAGCAGGTTACTCATCAGGACATTCCTTTATCTCTGGGCTAAGGTGGGAATGTACTTAGCTCTCATAGAEdit
Twelve nucleotide differences between the a and b alleles have been identified. This assay is designed to detect a T to C point mutation (indicated in red), however, additional nucleotide differences in close proximity have been indicated in green.
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
12964 | Fluorophore | AAC GTT AGT CTC TTG GCC TGA GC | ||||
12965 | Fluorophore | CTT CTC CAT GAG ATG CGT GTT C | ||||
12966 | TCT CTT GGC CTG AGC |
Component | Final Concentration |
---|---|
ddH2O | |
Kapa 2G HS buffer | 1.00 |
MgCl2 | 2.00 |
dNTPS-kapa | 0.20 |
12964 | 0.50 |
12965 | 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 |