Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr19:42031763+42031872 110bp GTCCCAGTGCCCTAAATACA CACAGGTTCAGCATCTTGGA
Mutant= 100 bp
Wild Type = 110 bp
Wt sequence with deletion in lower case
CTCTTTCCTGCTATTCAGAAAGTGTCCCAGTGCCCTAAATACATAGGGACTGGTAGAGTGAAGTCCCTGATAggccagggccagagagggggtcagttacagcttaagtggcttccaagatgctgaacctgtgcacctgtcttacctaggtcgcaatgagcccttgaagaaggagcggctcaagtggaagagtgactatcccatgactgatgggcagctgcggagcaaacgagatgagttctgggacacagcacctgcctttgagggccgcaaggagatctgggatgccctcaaggctgctgcctatgctgctgaagctaatgaccatgagctggctcaggccatcctggatggagccagcatcaccctgcctcatggtaagcaggcagggcctgggctctgtccttacctggactggggctggaggtttcattagcgcagaggtggaaagcaagtttctcatgtgagaagtttctgttttagctggtggagatgaaggagtatgcctatatgtgtgactgtcttcttcgtttaaatgactctttatggcatgaacaaagaagcacaaggcacagaaccaagcgtagtggcacatgcctgtggtcatgagaggtagacacagggggagctgaagttcaagttcaagaccagcctagattacatgagactgtcagaaggaggaaaggaaggagggagggatggacagaatccccctttgcctagtgagaagacaggaagacaccaataaaacctacaagtttaaaaggcactcagggcctgagagcatttgaagcactgatctggaaccttctgctgcattgccgcatttctgttcctagcaccctcattagctgtgaagggaaagtgacagtgaccttcagtcacagacctgggtctttttttgtttgtttttgttttgtttttcaagatagggcttctctgtatagccctggctgtcctggaactcactctgtagaccaggctgccctcgaactcagaaatccacctgcctctgcctccggagtgctgggatcaaaggcgtgcgccaccatgcctggcttcagacctgggtcttgactgtgttcctttctcagcttgtgacttttcctagccatatgtcccagagcagggcaagtctcaggttagagcctgagtctcttgcaatcacaaaggattaatcgtgatgtgcatagtaccacctagtctagtaccagcacttacaaggatactgaagactaaataaatgtgtttccacctgcattatatcataccagtaaggagacagggacccaacccaacacttgcatgtgcattcagccaccacaatgttgaataaacacataagaggtagcatcctgtggctgaggagccagaacctgatgaccaggaggttagccccagtctactgactatggggaagggctagggacagtgaggtagctgtggtgagtgaattaacctgcttacactgttgtagcagaggaccacaggcaggctccttaaacagtagaagtttgtttctcacaatttgagagactggaaagtccaaggtcaaaggcccagctgtttagtcctgatgatgcttcttggtttttttgctgtttttacgtggtaggcgtgcagaagagaggactgggtgggttctctgtttttatttggtaggcatgcaggaagagaggactgctcgggttctctggggcttttccttgaaaggaagctggtcctgtcatttacccgtgtaatcttattccttccaaatatgccttaacatgggagtttgggggacacagcattcagtgtgtgacatcaggccccccagactacactgatcaccctgcctttcccacaggaactctttgtgaatgctatgacgagctgggtaaccgctatcaactgcccatctattgcctgtccccacctgtgaacttgctgctggagcacacggaggaggagagcctggagcctcccgagcccacccctagtgtgcgccgtgaattcccactgaaagtgcgcctctccacaggcaaggacgtgaggctcaatgccagcctgcctgacacggtggggcaactcaagaggcagctgcacagccaggagggcattgagccatcctggcagagatggttcttctctgggaagctgcttacagaccgcacaaggctccaggaaacaaagattcaaaaagacttcgtcatccaggtcatcatcaaccagcccccaccaccccaggactgacggactgacggactgacggcaccaactcctaggaaaagaggccatggggggccctgcaggctgtggggcccagtcctgccgccttccagtgctgctgtcttcttcaggggcgcctcccaccgccctgctgtatggctcttagtgagctgcgaagggacttgccctctggggcactggatccaccagtgctcagcacccagggagcagtgctgcctgccacacacaggccttgccaaccttggagaaaaggcacatggcactctccaccacaggttggagccatgggggccgtcctggcgacctgtcatctggtgccacgctgtgctcactccagttttctgctcagggttggaagcaattgctgtctcccctttatccctcaccatgactctgccctgggcacagtgccagtcccctggatggggagggagcccggtgggccccagggcttaaggagcttcaggtgggcctctctgcctctccctgcacccagcacaatctgcagagatgaggcgggtggctggatagccaggcttcatggcaggggtttgcacagggctgtccccaactgtaacccaggcagggtggggtccgaagcctggtcatagcccagcggggccctgtgtacagacacatctgcattttatcaataaagcctttgctcctctcttgcctagcacatgacttcctgtttcacagcacgccagccagccggagcccagagcttctgctggcttggctgcagatgggcacagcagagcatccaactgatgctcctcctgGGGGCCCTAGTCTCATCCAACCTCGCTGTGTTCTGTGGGTCCTGAAAAAGGCCACTTCACCTCTGAACACAGTTTGCT
A 2945 bp deletion beginning at Chromosome 19 position 42,031,812 bp and ending after 42,034,756 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
44266 | GTC CCA GTG CCC TAA ATA CA | Common | A | |||
44267 | CAC AGG TTC AGC ATC TTG GA | Wild type Reverse | A | |||
44268 | CCT TTT TCA GGA CCC ACA GA | Mutant Reverse | A | |||
44269 | Fluorophore-1 | ATA GGC CAG GGC CAG AGA G | Quencher-1 | WT Probe | ||
44270 | Fluorophore-2 | CTG ATA GGG GCC CTA GTC TCA T | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
44266 | 0.40 uM |
44267 | 0.40 uM |
44268 | 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 |