Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
Mutant= 87 bp
Wild Type = 84 bp
Wt Sequence (deletions in lower case):
GCTGTTATCTCCGCAGGTTTCGGCATGACAACACCAGCcacagtgggcgggaaggccttcctcatcgcctacggactgttcggctgcgcggggaccatcttgttcttcaacctctttctagagcgcatcatctcgctgctggccttcatcatgcgcgcctgccgggagcgccagctgcgccgcagtggcctgctgcccgccaccttccgccgcggctcggcgctgtcggaggccgacagcctggcgggctggaagccctcggtgtaccacgtgctgctcatcctgggcctgttcgccgtgctgctggcctgttgcgcctcggccatgtacaccagcgtggaaggctgggactacgtggactcgctctacttctgcttcgtcaccttcagcaccatcggcttcggggacctggtgagcagccaacacgccgcctaccggaaccaggggctctaccgcctgggcaacttcctcttcatcctgctcggcgtgtgctgcatctactcgctcttcaacgtcatctccatcctcatcaagcaggtgctcaactggatgctgcgcaagctgagctgccgctgctgcacgcgctgctgcccggctcccggcgcgcccctagcccggcgcaacgccatcaccccgggctcccggctgcgccgtcgcctggccgcgctgggtgccgaccccgcgactcgcgacagcgatgccgagggccgccgcctgtcgggcgagctcatctctatgcgcgacctcacggcgtccaacaaggtgtcgctggcgctgctgcagaagcagctgtccgagacggccaacggctacccgcgcagcgtgtgcgtcaacacgcgacagaacggcttctcgggcggcgtgggcgcgctgggcatcatgaacaaccggctggcagagaccagcgcCTCCAGGTAGAGCGGCCGCCCGCCCCCAGCGTCGCGGACCGGGCCGGGGCTGCGCGCCGCCGGGCGGGTTTGCTTCACTTCTCTCCGAGATGGCGCTTTCTTAA
This mutation is a 870 bp deletion beginning at Chromosome 17 position 87,745,950 bp and ending after 87,746,819 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
62729 | GTT GAG CAC CTG CTT GAT GA | Wild type Reverse | A | |||
67017 | AAC TTC CTC TTC ATC CTG CTC | Wild type Forward | A | |||
67018 | Fluorophore-1 | CGT GTG CTG CAT CTA CTC GCT | Quencher-1 | WT Probe | ||
67019 | CAC ACC ACA TGC GAC CAC | Mutant Forward | A | |||
67020 | GCT CTA CCT GGA GGC TGG T | Mutant Reverse | A | |||
67021 | Fluorophore-2 | CCT GGC TGT TAT CTC CGC AG | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
62729 | 0.40 uM |
67017 | 0.40 uM |
67019 | 0.40 uM |
67020 | 0.40 uM |
Wt Probe | 0.15 uM |
Mutant Probe | 0.15 uM |
DNA |
Step | Temp °C | Time | Note |
---|---|---|---|
1 | 95.0 | -- | |
2 | 95.0 | -- | |
3 | 60.0 | -- | |
4 | -- | repeat steps 2-3 for 40 cycles | |
5 | 40.0 | -- | Forever |