Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
Mutant= 117 bp
Wild Type = 119 bp
Fam=Mut
Hex=Wt
chr3:96597447+96597565 119bp CCGGCTCCATCGGTGTTT TCCTCCCTCCAGAAGCAA
Wt Sequence (deletions in lower case):
TAGGCTTCACTACCGAGACTTCAGGCCTCTCCCAACCACCACCCCCATACTCCTAGATATCTTCGGCAGGTCCCAGTCCCGGCTCCATCGGTGTTTTTGCCCGAGCCGCAGCTATGCTGCACACCGAgggccacgctcttcttcgagctgtgggacagggtaagctacgcttggcccgtttgcttctggagggaggagcctacgtgaatgagggtgatgcccagggggagactgcgctaatggcggcctgtagggcccgttacgacgaccctcagaacaaggcgcgcatggtacgctatctcttggagcaaggcgcggatcccaacatcgcagaccgtttaggacgtaccgcgctgatgcatgcttgcgccgggggtgggggagccgcagtggcctctctgctccttgcccacggcgcagacccttcagttcgagatcacgctggcgcctcggcgcttgtccacgccctggaccgaggggaccgcgagacccttgccacactgctggacgcctgcaaggccaagggcacggaggtcatcatcataacgacggatacctcgccctcgggcaccaagaagaccaggcagtatcttaattccccaccgtccccaggggtagaggaccctgcacccgctcctcctagtccaggggtctgcacatcgccttcagaagtccaactgcagactgcaggaggagggcgcgggttgttatcccctcgcgcccaggaagaagaggagaaaagagacatttttgaatttcctcttcccaagccccctgatgacccttccccttccgagcctctccccaagccacctcgacatcctccaaaaccactcaaaaggctcaattccgagccctggggccttgtggctcctcctcaaccggttcctcctgcagaagggaggcccggtttggagcgtctggctgctgaattcaatggcttgaccctgaccgggagaccgcgtctttctcggcgtcacagcaccgaaggcccggaggacccgcccccatgggcggagaaagtgacgggtgggggtcctctctcgcgccgaaacactgcgcccgaggctcaggagtctggtcttccttcagggctaaggcaaaaactgagccgcatggagccggtggagctcgacacccctggacatttttgccctgactcccccgagtccagccggttatccctggagcgccggcgatacagcgcctctccgctgaccctccctccagcgggctcagtttcctccccacgccagtcccaggaaagtctgcccggggctgtatccccgctgagcggacggaggcggagtccagggctgctagagagaaggggctccgggaccttgctcctggaccacatctcgcaaacacgaccaggtttcttgcctcctctcaacgtcagtccccaccctcccatccccgacattcgtccccaacccggaggtcgggcaccctcactgcctgcccctccaccctctggggcaccagggtctcccaggaccaagcgcaagttggtgagacgccactccatgcagactgagcagattcgcctgctaggggggtttcagagtctaggtgggcctgGGGAACCCGGGCGCTGAAGGAAATGAGGGGCGCCAAGGAGGGGGAGGGTCAGTACCCTGATAGAGGGAGAACCAGCTGATACCTCGGACACGGGATCTCTTGCATGTGCTC
This mutation is a 1457 bp deletion beginning at Chromosome 3 position 96,597,496 bp and ending after 96,598,952 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
66521 | TCC TCC CTC CAG AAG CAA | Wild type Reverse | A | |||
66522 | CCG GCT CCA TCG GTG TTT | Common | A | |||
66523 | TCC CTC TAT CAG GGT ACT GAC C | Mutant Reverse | A | |||
66524 | Fluorophore-1 | CTG CAC ACC GAG GGA ACC C | Quencher-1 | MUT Probe | ||
66525 | Fluorophore-2 | TCG AAG AAG AGC GTG GCC | Quencher-2 | WT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
66521 | 0.40 uM |
66522 | 0.40 uM |
66523 | 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 |