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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 |