Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr10:75230953+75231047 95bp TTGGGGTTCACTCTCTGCT TTCGTACAGATAGACCAAAACACC
Mut = 102 bp
Wt = 95 bp
Fam = Mut
Hex = Wt
Wt Sequence (deletions in lower case):
CTAGCCAGCACATGGTATAAGGTGTTCTGTCTCCTGAATGGGTTAGGACACACATTATAAATCTGTAAAGTATTAGCTTCTATAAGTGAACTGAAAGATTGCCAACATTTTTGTTTTGTTATTGAGACATCTGTCTTTCAGATACTTTTAtcatgtcgtggaaattttcagaaataatttctaaagagctgctttgtcgttttcttttcaacatgatgcctcttctattcttcccacaggtttggttgtaaatgcatcaccaagaggcagcccagaatgaagaaagcaaacaggagtgctggctcagtgcctaaggtgtctgggataagcaaaccgcaaacagtagagaaaagcaagcctgagaacagctcttcagcacccacaggagtcaagcctgtgaggcctggggcagcagcagccttgtcaaaggtattcacgtagctgtatttgaatatgcagtggattttaggcatgtatttaaatatgttgaatttattaaatgaatataatttggggttcactctctgctagtactaggaacacagtctttgaggagatctttcagcagGGTAACCTTAATTGGTGTTTTGGTCTATCTGTACGAATGAAACTTAACAGTAGGCTCTGTATAAAGAAAATAACCAAGTGCCTGCTTTATACAGGGAATCATGTACAGGTGAGCCAGAGGTAGAGTCTGCCTTCAGGGAGTTTGAGA
This mutation is a 408 bp deletion beginning at Chromosome 10 position 75,230,598 bp and ending after 75,231,005 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
49320 | TTC GTA CAG ATA GAC CAA AAC ACC | Common | A | |||
49321 | TTG GGG TTC ACT CTC TGC T | Wild type Forward | A | |||
49322 | GTG AAC TGA AAG ATT GCC AAC A | Mutant Forward | A | |||
49323 | Fluorophore-1 | TAC TAG GAA CAC AGT CTT TGA GGA GAT CT | Quencher-1 | WT Probe | ||
49324 | Fluorophore-2 | TGT TTT GTT ATT GAG ACA TCT GTC TTT CA | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
49320 | 0.40 uM |
49321 | 0.40 uM |
49322 | 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 |