Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr8:77555755+77555864 110bp TTGGAGCTGTTTCCTGATGA CTTGCTTTGGCCTGATAGAGA
Mutant= 129 bp
Wild Type = 110 bp
Wt Sequence:ACTGCTGGACTGTCTCTGTAGCCCCTTAGTTGTTTTTATTTATTTGCTTATTTTTGCTGTTACCatgagccaaacccaggaccttaagcatcctaggaagtcctcattcacgtttgttgaacagtacttactaaaggttaccagttggaggccatcattcatttcaggctcagttccacttgctggagtcttcagctagagttgcgtattcctcattcttttttactaatttgtacttttttcttgtttataaaggaaacttttcagtatacacttttcaagtgggctgaagagcttcatgcactgagccgaatacaagacttacttggttgctatgagcaggccttggagctgtttcctgatgatgaagtgatttgcaacagtatgggagagcacctcttcaggtttgtcatgacctcATGAGTATAGGATTATCTCTATCAGGCCAAAGCAAGGATAAATCATGCATTAACTTACTTGGTTTGGTTTTGAGATAGAGTCTTACCACCAGCTCAGTCCAGTCTGGAACTTGTAGGCTTACATCTTGTTTTAG
Deleted Sequence: atgagccaaacccaggaccttaagcatcctaggaagtcctcattcacgtttgttgaacagtacttactaaaggttaccagttggaggccatcattcatttcaggctcagttccacttgctggagtcttcagctagagttgcgtattcctcattcttttttactaatttgtacttttttcttgtttataaaggaaacttttcagtatacacttttcaagtgggctgaagagcttcatgcactgagccgaatacaagacttacttggttgctatgagcaggccttggagctgtttcctgatgatgaagtgatttgcaacagtatgggagagcacctcttcaggtttgtcatgacctc
355 bp deletion beginning at Chromosome 8 positive strand position 77,555,474 bp and ending after 77,555,828 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
37473 | TTG GAG CTG TTT CCT GAT GA | Wild type Forward | A | |||
37474 | CTT GCT TTG GCC TGA TAG AGA | Common | A | |||
37475 | ACT GAA CAC CAA GAG CAG CA | Mutant Forward | A | |||
37476 | Fluorophore-1 | TGC AAC AGT ATG GGA GAG CA | Quencher-1 | WT Probe | ||
37477 | Fluorophore-2 | CTC TTA ACT GCT GGA CTG TCT CTG | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
37473 | 0.40 uM |
37474 | 0.40 uM |
37475 | 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 |