Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr12:56339872-56340000 129bp CAAGTGAAGGCCGAACTCT AAAACTGCTCCCTTGTGAGG
Mut= 118 bp
Wt= 129 bp
Fam=Mut
Hex=Wt
Wt Sequence (deletion in lower case; and insertion with carrots ^g^ (G insertion):
TTAGGCATGTGTTTGTGTGATATGTAACAAAGAGAATACACTTTCTGGACTGTCGTAGTTGATCTTCGTCTGTTGAATGCCTAGTTGGGATTCAGTAGGATTCTGACTAGGGCAGATCTATTTTTGGATACCTTCCAGTGGTCTTTAAAATATCAAAGTATCAAAAGAGGTTTATCTTTCATGGATGGCTTTTCAAAGATGGCCCCAGGAACAGAATTAAAATAATAAGACATGTTTTTACTTTTTATTTTCAAAACTGTGCCTTTTGGCTTTTTAAAATGGCAACTGGGTAAAGCATTTTATTATGTCATTGAAACTTAGTAACTTTGAGCAA^aataggcatgcattactccatagaaatcttatttttgtgataatcccagatatacagcttaatcgtgtaatttttgtgatgttgctggtttgcctctttgaagcttaacctcagagatgatgtggtgaaaattacaatcgattggaacaggctccagagcctctcagcatcccagcctgcactgctccttactgcacttgaacaacacgttttgtatttacaggtaaatttcatattagaaatatttactggatattgaaaatagaaattacctatagttaaattaactggagtatttttcagttctacctcaacattctgttaacagtattattaataaaaggttattaaaatccatattgagtctaggtctgttaatttcactagtgatcggaatagttatgagaatatagcccaataggccaatgtgggtatgagattgaataaatgcaattctattcaattctaggctttgctgaaaattcaatacagttcatctatggtgggttataattaagcaataagacacggcaggtgtgagtcatgctatgataatgattccatgccttgggtgagtttggaggctcatggagtgctttcagtggttcaagaagtggcattatcccagcatgatacatcctggagtgtctattgcaattaaacagtttcagcatagtttttaaaaagaaagtaatttctgtgacattaatgtttcatcttagtgagtagccagtcactatttcatcttaataatttgaaataatttatcctttattggaagtcaatattgaattatttataatggtattgttacatttttaatatttcagtttaagaaatgaacagtattataaccttgctgttgttgtttaacatctctgcgagtgtaattgatgggagttggaggttttctctcatcaggataagcagaactgctttttctggtcactttaccctgttgaagatgttagaagatttcagaagagtactttctgggatgtttagctgtgtttcatgataaaaataacagttaacactttgtttaaagggcaagtgatgagctatagaactcaaatgtggaccatatccaggtggggatgacaggaaaggaagtgaatttccattatgaagacattttttttaacctactgtagttggaatgatctgtttccaacgtgtatagcatgttttgttttctttctttctatctacctatcttccttccttccttccttccttccttccttccttcctttctctttcttctccccgtgtgcattttcttttgtgaaatttcatttcatgtcccaataagatatcatactatgacagagaaaggctgactattcactcagggtaacacctatatacttcatatccatgctctctcattcattttggccaactgtactttgaagtggagagtctcagcgatacagtgataatgcagggctttgctgagtgagcactgccctcctgctaaggagggagaacactcagcctagtgttctgaatgacatgttaattaagaggcatccaggccagagtctgaggctttgatttagcacaaaataaaagatgaatgacagaatgatttcctcaaagaaaaatttaaatgtcctcaggttttctttcttttcttcttttttaaaaataaatgtacagtaaatgataagggcaatgtacagtagtagaaaacagcaaacctgtgtgtgttaagatctgtgtgtccttgtatgccagctttttatgtttaataactgataatattcatttctgtagcagcagcttagataaatctatcactagctgagatgagtttgaacctgaacagatgtaactgtgttctttccagccttttttagcaaaacttcagtctttaatgaaagaaaactcaacagctacagagataagacagacagaagcagagaccaagagtgaactaagagccatacatcccaccgaggacctacaagatgaagggaagcccaaagactgtgatgtaggagatgtgaaaaagacacaaaacctttttgacccagaagtagttcagatcaaggctggaaaagcagaagtgagtgtggacaggtacacgggtcagggtaacattactttgaaaagttctgttgtgtttagattgttaaattatttgttgttttagattgacagacgaatatctgcatttattgagagaaaacaagctgaaatcaatgaaaacaatgtcagggaattttgcaatgttattgattgtaatcaaggtaactacaagtttttttttttaacagaattttaaacatcttgacaactgctataatattttgttttaaaaacatataacattaagaacaagattctattttgcaggtgtttagttgataattaaggtagtagaattttcctttatttagtaatgggctctgttgccaaactgattctcattttggttacataaaaaatccaagtgaaggccgaactctagagacagatgtttaccacaggttcaagaagatcagagttcctttagaaa^GTGAAGGTGAACCAGCGAACCTAGTAGCACTACTTGCACCCCTCACAAGGGAGCAGTTTTCTCCTGACTCCTGAGTTTCATGGTGGATACACCCAGCTTGCCATGAAGTCCTGTGGAGCCTCAGCCCCAGGCCTCTCCCTTTTCACACTGCTGTCTCACCTTTGCCGTTTCGCCTCTGTCTCCCTTTCCTCTTCGTTTGGGGCTACAGTGTTTCCAGGTCTGCAGAGGACCTGCAGAACCCCATAGACTGTGTAGATTGCCACAGATCATAACCAGTTAGCTTTGTAATAACCAGACTGTAAG
This mutation is a 2527 bp deletion beginning at Chromosome 12 position 56,339,932 bp and ending after 56,342,458 bp (GRCm38/mm10). In addition, there is a 1 bp insertion (G) at the deletion site.
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
48409 | CAA GTG AAG GCC GAA CTC T | Wild type Forward | A | |||
48410 | AAA ACT GCT CCC TTG TGA GG | Common | A | |||
48411 | AAT GGC AAC TGG GTA AAG CA | Mutant Forward | A | |||
48412 | Fluorophore-1 | CCA CAG GTT CAA GAA GAT CAG AGT | Quencher-1 | WT Probe | ||
48413 | Fluorophore-2 | AAC TTT GAG CAA GGT GAA GGT G | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
48409 | 0.40 uM |
48410 | 0.40 uM |
48411 | 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 |