Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr5:53276821+53276927 107bp TTGTGCCTGTCCCTTCTTTG TCTCAGCATTTTGGAAAGGAC
Mut= 103 bp
Wt= 107 bp
Fam=Mut
Hex=Wt
Wt Sequence (deletion in lower case; and insertion with carrots ^g^ (TG insertion):
ATGCTTGATTTCTCTTGGCCTCTGCCTGTTGTGCATCTCCAAAGTGCCAGGCTTAGCTGCTGCAGCTACAGCAAGCTCACTAATTGGCACAGCACCTGCAATCAATTAGGGAAGCTTCAATTATCCAAACCAAAGCCACCCACCCAAAGATTAATTTTCTGCTCTATTTCCTATCAAAAACATAGCATATTTAGCTTGGAGTCCAGTCCTTGAGTGGAGTGTTTTTAGCCACCGTTTTTCTGATAATTGTGCCTGTCCCTTCTTTGCGGCAGCTCTCCTCT^tgtaatgtcacacccaccagctctttgaaacagttctttctgttgtgttgtgtcctttccaaaatgctgagatcacaggaaaaagatgttggttgggatctttttgtttgaagtgtggctctttttacatctaggatctagcttctataacctaaaagctttaagtaatgacgtccctgaattgaatcacactcctaaatatgcttgtaattagcccatataaatagaagcaaaatgcccacatctcctttctaaagagtaattatttgttctgttgtgtttctcagagaaggagcaggctgtaaatcgagctggtattgtccaggaagatgtgcaaccgccaggtaaactgaaaacgctcagacaatcccttccggggcacgtggatgagtactttgctgtggagtgggtggttcttctccctggaacctgatactgttcattattttatgatagttcctctcttgtcagggctatctaagacttcatgctaatatttcagtgcctagtactttttacaagcttcctgggcccctcgtttgtccttggtaagtggacataaaatggaatcaagtatgcaacagatcatctgctttgcagactcccatactgagcacccctgtaaatacctccctcactggcagaccgtgtgtgagggtgagcactaaggttcagtttcctagagaagttctcaaagctcccttcagggacgtgtgggatgttcctttaaaatgatacaagttggctgagtacgagagattttctagcttattaaccaaactcctgcagtctggtggtcctaggctagatccatttcttcctagcagggaagagttcctaagcctttaaaaggatacctcatagctgccatggctgcctcgttgcagggttccagtggctgcactcacccatttcaatgtctcagccacatggaaattgcatggctcaatttccacatcatcttgtttgtttgggatcaagggggtgtgttcagtcatttagcaatgaatcctgatagggtatttgtgtttctttccatagggttgaaagtgtggtctgatccatttggcaggaaatgaggctgtcagcaagtctgatgaggaaagtggacgtctttatcctgtgcactccgcagtggggacaatagatgcctcactgtggcagcatggcatggagagggaactctcatgctgctagccagaccccttgtgatagagactgtgtgcaaagacagtgcttcccttaactccctggagaacctgaacagatgccaccattaggaagtgccttgcggctccattgactttgcaggagcagagccagcctgcaaggctgtttgtggaagatctgctgctcctgcagtctttatcacttccaagctgtgatgtgaacacaagcaacctgtgggctcaaggtccgtggctgctctgacaccttctgaataagcgatttcagtgcaaatggccttgccaagctgcctcgcagggctcttggaggatgtttcagttgataaaactgtttgaagacaggatccttggcactgtttaagaatatacactgctcagcttaaccatctcattgaaagtcactgtgtgtggaagtgaatagggagcgagtcacactagactataccacacacagtagactcctgcgtgaggctgcaggtattaaaatggtttctcttagtttgctggtgtgtactgtttttatattttgtgtgtgtatatgtttgttttatttggatgggtggtgtttgttggctttggattttgttttttgagacagggtctgtcagaccagtcaagccttgaactcccagagatccacctgtcactgcccagtagaatttttttgtaaattgaattctgatgtgaagaagtagaagtagtgctattgtctagattctatttgagtagatatccgtgtagt^AATAGCTATTTGTAGACAAGGTGAGCGGCACACTTCTTGTGGCAGCTGTGGCATTAGGTTCTCCAGAGAAACAGAACTGATGAGATGAATATGATTTACATCAGTGACTTACAGGCTGTGGTTCACCTAATCCAACCATGCCTGTCTTATCAATGGAAAGTCCAAGAATCCAATAGTTGTTCAGTCCAAAAGGCTGGACTTTAGTATCTACCAGAATCCCGAAAAAGTCGTCTGTAATCCCCGTGAGAGCAACGGCAAGCAGGCAGAGAGCACACA
This mutation is a 1929 bp deletion beginning at Chromosome 5 position 53,276,856 bp and ending after 53,278,784 bp (GRCm38/mm10). In addition, there is a 2 bp TG insertion at the deletion site.
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
48494 | TTG TGC CTG TCC CTT CTT TG | Common | A | |||
48495 | TCT CAG CAT TTT GGA AAG GAC | Wild type Reverse | A | |||
48496 | CTG GAG AAC CTA ATG CCA CAG | Mutant Reverse | A | |||
48497 | Fluorophore-1 | AAT GTC ACA CCC ACC AGC TC | Quencher-1 | WT Probe | ||
48498 | Fluorophore-2 | ACA AGG TGA GCG GCA CAC | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
48494 | 0.40 uM |
48495 | 0.40 uM |
48496 | 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 |