Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr1:34473174+34473298 125bp TAACCCAAGGCCCCAAAGA TGCTTTTAAGAGTTCCAGGGAC
Mut= 140 bp
Wt= 125 bp
Fam=Mut
Hex=Wt
Wt Sequence (deletion in lower case and insertions with carrots ^g^ (GCACCCATTGCA insertion) ):
GGTAGAGACAAGTGAACCCATTGAGGGGTTGCCAGTCAGTCAGGCAAGCAAATCTGTGAGCAGCCTGTTTAGTAAGAGATCCTTTCTTAAATTTAATATTGACCTTTGGTCTCCACACTGTCAACACACACACACACACACACACACAAAATGGGTGCATACTAATGTACAAAAAACCCCAAAAGGTTTTATTTAAAGTTTTAATCTAGGGATCCTGCAATT^taagaggtactgatctccttcaggtcagctcagaaggggttgtggtgtctgtacagttaggcagtcctgacatcccagcacttaggaagctgaagtcagaggctagcctgggcaacctagcacaagacctgtgggaaaaggaggaggaacagaatggggaaaggggcattgtgggtaggctatgatggtgtctgctctgattcacgattgtcagtgctggctgcggacgaacaggtgtcctgtgcgctgttgactatgtgaggcagttgctgctgacccaggtatgacgtcctctgctctgtgtgcagtatgtcccatgtctgcctaccagggtctggtcacacttcccctctctatcccagacaatccctcccaacttcagtctcttccaagtggtcctggagatgcggaaacagcggcctgcagcagtgcagacagaggtatgtgcttgccccgtcttcctgttcctaaggacgagggaggtttgcttagcttcccaatctccccttctcaatgaccactcaccccaacaggagcagtacaggttcctgtaccacacagtggctcagctattctcccgcactctccaggacaccagcccccactaccagaacctcaaggaggtacagagcccccaatttcaccctttctgccagccatcaacacctatacccttcttgggaacagcaaccgacttactgtgaaaggcacctttgcctcctcagagctccttccccaggctgttccctaagctcacctaggcacccacagtgacctgcagcagggctatggcctcaggaccgaccctgcctgcttccctgcagaactgcgctccaatctgcaaggaagccttctccctcaggacctcctcagccctgcctgccacatcccggccaccaggaggggttctcaggtactgggctcactctgtccctggaaaatcaacctctacagtctgggttagcttttcccttggtcttccagaaggtaattaattaggcaagtacatttttaaagaatttcaatctctctctctctctctctctctctctctctctccctctctctctctctctctctctctctctctctgtgtgtgtgtgtgtgtgtgagagagaaagagtatgtgagtccttaccttccacgtgttttgagaaacagtctcactgtgtacatcagagcttctggggactcacctgtctctgtctcattgtaggagggttggggtctcacacactctaccacatgcgtcctccctagtcccacttgccaatctggccctgtccctcctgctgtggggattctagggacacggaacctctactttctgccctcaggtggcccgccccttccctcctttcctgctctgaagactggggcaggcaggaaggaagcccgtagcctgggctgagcttgctcccccccccccgcccccctcccttgtgctaagcgctttcacttcctctgcttgttgcccgctgcaggagcatctcggtgcctgcgcccccgaccctccccatggctgacacttacgctgtggtgcagaagcgtggcgcttcggcgggcacagggccggggccgcgggcgcccaccagcacggacaccccgatctacagccaggtggctccacgtgcccagcgaccggtggcacacacggaggacgcacaggggacaacggcactgcgccgaggtgagttgaggccctccgggggtgtggagctgaggacaagtacctattagcaactttcccgctctcctaaggctcccgagggtctaagcgctgctcggaggtcagccccagatccagcatggccacgcccctaggctactaaacgtcggggttcaagccccaaaatgagccccaggtcaggtctcaattgggagactgggctgtctaacccaaggccccaaagacagacctccgaacttggacacgcccatggacacgcctcctaccgctacctcgggaagg^GAAATCGGTAGCTATGCTGCTCTAGAGTCCCTGGAACTCTTAAAAGCAGCTTGAGCTGACCCATAGGGGTTCTCCTGCCCTCAGTTCCTGCGGACCAAAACTCTTCCGGGCCTGATGCCTACGAAGAAGTAACAGATGGAGCACAGACTGGAGGGCTAGGTAAGTCAGAGCCTAGGGTGCAGCTTTGGAGAGCCCCAGGACTTCGGGAGTTCGGGCTGGGGC
This mutation is a 1972 bp deletion beginning at Chromosome 1 position 34,471,279 bp and ending after 34,473,250 bp (GRCm38/mm10). In addition, there is a 12 bp (GCACCCATTGCA) insertion at the deletion site
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
52772 | TAA CCC AAG GCC CCA AAG A | Wild type Forward | A | |||
52773 | TGC TTT TAA GAG TTC CAG GGA C | Common | A | |||
52774 | CAC ACA AAA TGG GTG CAT ACT | Mutant Forward | A | |||
52775 | Fluorophore-1 | AGA CCT CCG AAC TTG GAC ACG | Quencher-1 | WT Probe | ||
52777 | Fluorophore-2 | CTG CAA TTG CAC CCA TTG C | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
52772 | 0.40 uM |
52773 | 0.40 uM |
52774 | 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 |