Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
Mutant= 121 bp
Wild Type = 141 bp
>chr7:99626189-99626329 141bp ACATCGAGGTAGTAGAGGAC CGTGATTGAGCTGTAGTGAG
Wt Sequence (deletion in lower case):
AGCTGGAGGACGGACCGGTCGGAACTGTCAGCTGCTCCGGGTCAAGGTCTAGCCCACAGCCACCTTCCCGTGCCACCCCCATCCCATCATCTCCAAACGCTCCCAATTCACTGGTGAGCGCTGCGGGCCGGGGGCTGGATGCGGGGACGGCCGCGATGGCCCCGCGCGCGGgacagcgggggctctggagcccgctgccagggctgctgctcttggcggcggcgctgagccggcccgccgcgccctgtcccttccagtgttactgcttcggcagcccccggttaatgttgcgctgcgcgtcgggcgcggagctccggcagccgccccgggacgtgccacccgacgcgcgcaacctcaccatcgtgggcgccaacctgaccgtgctgcgcgccgcagccttcgcgggagggggcgagggggcgacggacggcgtgcgcctaccgctccttaccgcgctgcgcctcacacacaacaacatcgaggtagtagaggacggtgccttcgacgggttgcccagcctggcggcactcgacctaagccacaacccgctacgcgccctgggctaccgcgcctttcgcgggctgcctgcgctgcgctcactacagctcaatcacgcgctggctcgaggcagccccgggatgctggatgcactggacgccgctctggcccccctggccgagcttcgcctgctgggcttggtaggcaacgcgctgagccgcctgccactcgccgcgctgcgcctgccccgcctggagcagctggatgcgcgtgtcaacgcgctggccggcctgggcccggacgagctgagcgcgcttgagcgcgatggcgacctgccccagccgcgcctgctgctggcggacaacccactgagctgtgggtgcacctcgcgccccctgctggcctggctgcacaacgccaccgagcgcgtacccgacgcgcgccgcctgcgctgcgcttccccgcgcgtactgttggaccggcctctgatagacctggacgaggcgcgactaggttgctccgacggcgatgcacacgagagcggggaagggatagacgtcgccggcccggagttggaagcctcttacgtcttcttcgggctggtgctggcactcatcggcctcatcttcctcatggtgctctacctaaaccgccgcggcatccaacgctggatgcacaatttgcgcgaggcctgcagagatcagatggagggctaccactaccgctacgagcaggatgcagacccacgccgcgcgcctgctccggccgcccctgccggctcccgggccacttctccaggctcggGTCTCTGAGCATCACCTCCCTAGTGGGAGGCTGTTCCTATCAGCTGATGACTTTACTTGGGCCATGCAGCCTTTCTCTGCCTGGCCTGGGCCCCGGAGATAACACACTTACGAGTTTGGATCTCTGAGACCTTTGGATCAATCGTAGTATCTCTCCCATACAGAAATTCCAAGGTGTACTCTTTCCCTGGCCTTAGAGTTTCCAGATCAAAAACCCAGAAATCTGTTTTCAGAAACAAATCCTGCCTGCCGGTCCTTA
This mutation is a 1131 bp deletion beginning at Chromosome 7 position 99,625,502 bp and ending after 99,626,632 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
69011 | ACA TCG AGG TAG TAG AGG AC | Wild type Forward | A | |||
69012 | CGT GAT TGA GCT GTA GTG AG | Wild type Reverse | A | |||
69013 | CCC AAT TCA CTG GTG AGC | Mutant Forward | A | |||
69014 | GTC ATC AGC TGA TAG GAA CA | Mutant Reverse | A | |||
69015 | Fluorophore-1 | ACT CGA CCT AAG CCA CAA | Quencher-1 | WT Probe | ||
69016 | Fluorophore-2 | CGG GTC TCT GAG CAT CAC CT | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
69011 | 0.40 uM |
69012 | 0.40 uM |
69013 | 0.40 uM |
69014 | 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 |