Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
Mutant= 120 bp
Wild Type = 114 bp
chr2:157556069-157556182 114bp CAGGCATTAACAGGAGGGAGT GGGATCCACTCCTCAAGTCC
The Wt For primer (primer 59969) anneals over the nucleotide sequence containing mouse genomic variations rs27338091.
Wt Sequence (deletions in lower case): GGCTGCAGCCCCAGGAGGCGATACATGATCTCTGCTGGGCAGGGCCCAGGCAAAGCCAGCCCACTGTGGGGACCGTGTCCTCCTGGGATCCCCCAGTAGAAGCctagggaagacacaccctagcaggctatctttaagtaataatttggataggagtagacattcatgggtgggtggggaggaaagggcattcacatttgctgttgttaaccatgatccattttcccaaataagtatacaatttgttcctgtttttttgtttgttttgtttgttgttttttgttttttgtgtgtgtctgtctgtcctttgtctgtttgttgtgttgacttttctcttcaggatccctgctgccttggtgatcccgggctgacagccagagagcacagcggctcagctcctggagagagagtcgaagaaagcgaagggcagccacctgtgcctgctggctcccattaggtcggttcctgcagcggtgcctggcagccttggtgaaggccctgcccggcagagatcatgtattgcctccagtggctgctgcccgtcctcctcatccccaagcccctcaaccccgctctgtggttcagccactccatgttcatgggcttctacctgctcagcttcctcctggaacggaaaccttgcacaatatgtgccttggttttcctggcagccctctttctcatctgctatagctgctggggaaattgtttcctgtaccactgctccgattccccacttccggaatcagcccatgaccccggtgttgtgggcacctaacgtctgccgagatagcttgccaaggaagcagaagacgggaggggaggcattgacataggtcataaagcattggagtttcaaatcccgcagtcctgcgggtaccacattcctaatggagccttttggcctgtgatgttttatccttacaatgtgaataatggcactgatcggtgcttctgttgtagagtcctgtagtcgtgggtggtcttatggttgtgtgttctgtcaccatctgggtcccggctgacggattggcccacccccttgctcattgatttggggaatctatacccttgatatgacctgtgtggatacagtgtagtctcaatgtcacctccataacccttcctcgtcaagaccttcctcgttccctcctcgtcccctttccccgttttccccttggttcacttccaacccctttccttttttggggagcacctgtccaagacagggcttgtttttgcacttatctcaaatttgaagagattgctgatgcccgagagcctcgctttttcatccttcgttcccttttgagaaggtgagacggaatcatgtctcaactgctcgttgtctgcagacctccagtatttcctctgcctcatttttaagaaagaagcgatggggagacattgctctttggcctgggtatctgggctcctgccttccagcccagcctctctccctttgctcttcctcctgtctctctcagctgacctaaaggggccacctcatgtctccagtgcatgctcttcaggagggagatgtgcagtattctcgtagacccagtggtccctggctgagtgaatgagaaagtattacatttttcatagcagccatgattcccttggtaggtgtttggatatttttgatgtgccctgtatgtatgtgtgcactagtgtcagcgtgtatgtacacacgtatgtgtgaatgtgtgttgtgtgtgtgtttacataccaatacatgtgtatattccttttgaagaagctttattgaatatgttctgattttgaggtttagtagtagtagctagctgtagtaggtcctgctgcagtttttatttagcatggggattgcagagcgaccagcacagtggactccaaggtggttcagacaagacccaggggagcagtcgccatcatcctcccaccaggagcttcctcattgctgcgcacgtagactgtacactatgaagaaaacacaggaagaaagatttggtgacttggtacttgtttgcttttctctgcgcttcagaaacaagtgtttgcaaatgagactttctcctggcccctacccactggggatcagcatggttgttcttccagtcggaaatgtacccccctcctttccccctcttgtgtgcaagtggggggggggggcaggcataggacagagctggaagcaggcttctggggagtgggacttagaggccacacttgtgaaacactcggactgctgttgtaaagcttttatttctggtgtgttcgttccacagctgtttgaaatgtttaataaagctttataaactttactttgtggttttatgtggctgcagtccatttgggtcatggtgttggatttctgagttggtggggggcagggtgaagcacacagggctgagtccttggcgccagagtaccttagctggaaaatctgatctgttctctggagaacagatgctggaattggggaggaaagcagggggcagctcaggcattaacaggagggagtctggaagttttcagagatgccatttgcacttctcagctcccatggcttcctgcctgaagcTGTCTGCTCTATAGGACTTGAGGAGTGGATCCCTCGATAAACAACTTGGGACAGCTGGGG
This is a 2463 bp deletion beginning at Chromosome 2 position 157,398,022 bp and ending after 157,400,484 bp (GRCm39/mm39).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
59969 | CAG GCA TTA ACA GGA GGG AGT | Wild type Forward | A | |||
59970 | GGG ATC CAC TCC TCA AGT CC | Common | A | |||
59971 | GGC GAT ACA TGA TCT CTG CTG | Mutant Forward | A | |||
59972 | Fluorophore-1 | ATC CCC CAG TAG AAG CTG TCT G | Quencher-1 | MUT Probe | ||
59973 | Fluorophore-2 | AGC TCC CAT GGC TTC CTG | Quencher-2 | WT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
59969 | 0.40 uM |
59970 | 0.40 uM |
59971 | 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 |