Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr10:85927489+85927598 110bp AGGAAAATACTTCGTGCTATGG GTTATTTCTCGGAGTGCTTACTGT
Mut= 97 bp
Wt= 110 bp
Fam=Mut
Hex=Wt
Wt Sequence (deletion in lower case):
AAGCCTTCAAAGCCAAATCCTTTCTCCTTGGGATTGGGGCTCTCTTCTTTTCAAATGTTGTTTTCCAGTCCTCTGGGGAGGAAATAAACTTAATGATGAGTACAATAGGAAAATACTTCGTGCTATGGGAAGATATTGTTATATCCTGAGTGggattagaagaggttgggaggcaggactaaaggtgtgtttacagtaagcactccgagaaataactggctaaaagtgtttgttactgttcgggcttttgatgtgtttttacagagcagcagaagcacaaagggccccgcgttaaacataccttagaacacaatgtgcggatccttacattcgatgctaaagtacatatattttgttcttgattcctttgaaaagtgtctaatttggtttttaaagattctatttcgcagaagaaaacctcgggcttccccggaggtccaaggaggacttgacacatcgtctaaccacaagagagcagtcttccctttgatgagcgcttcttctccacctccccagaaatgaaagagcagagaggtatgtttgcaaacactctgaaagctctttccatcagacctgaacgccggaaaccacaaagggcagtcaagtttggattttcaaggagaacgccggagtaaactttcgctctgttggccagccgatcagccagtgcctggggaggctctctgctcaattcctgaacaagttcgggctcccggatagccacacttgctgacccaaggagtgaattacaacaattgggcagctttgaaagatcaatctgcttagagtgcgaccgtctatccaggctgggaaccctatgttaactcttaaactaagatcagaggaagcacatggatacaagcgttgatgaggcgagccctctatctttgccccttcttctctgaagctttataaagacccaagaagagcaacctgcttcggaaatcatgcaaagttcactccaagtgtgctcaggtacctaacttctggacctagaaacttctccagagacagaccgggaagtctggatggaccagaatctccgctacgacagcttccagtctcgtcttcaaggtgagagagtcatctgcgtgctttttcgcagagaatggaaaaacgcagatcggccggactgcttgccctgccgtccgcgctacgtacagcgccgctgggcgcgctgcccgggagggagccctgcagggtctcggtgcgccaggatgccgcggactgcgcgcggagacggccgtactcgtccctgccgcttgggggcgtcgccgagcccgcctttttgcgccaacgcaacgagcgcgagcggcagcgcgtgcgctgtgtgaacgagggctacgcgcgcctccgccagcacctgccgcgcgagctggcgggccagcggctcagcaaggtggagacgctgcgcgccgccatcagctacatcaagcaactgcaggagctgctggagcgccaccggccggactgcaacagcgatggcgagtccaaagcatcctccggagcctcgccctgcagcgagtctgaggagcgtagctagcagcatcctagcccactgggctttcatggttgcttctccgggcctagatctttaaaacaaaaccaaaaaccagaaaacaactaagtgcttctggatctaggaaagaaaggtaattgaatgtttagagagtttggttttttttttttttttttttttttgtcccctttcaccttcattggttccgagtttgcagcttaaaaatgcttaaggaaactcaattctcgtatttttgaagcacagaagggccatctcaaatctatgcggtgattttaaaaacacaaaggactttaagaaaccttagccagagaagaaacacacacacacacacacacacacacacacacacacgagacagtccttcacgcgagaggaacattctcactcattccttccaattgggaaatgcactggaaatctcaacacctcactgagggtaggacactccataagacatttgtggagcatctgctggacctgaacgtcgctgtgctaaaaaggacaatgcttgacctatcttgcttcgtccgcaagaggtagatcggaaactttagcagctaataaagtgccgcgaagttaatgatctttttggaggagctctgaatggccatcttctctcccggtttctgcccactgtttctcttgcccttttgtctatgtgttcccctgtacagttctcatcccgtctaacctttgcttggaaaaaataaaaaagcaagtatgcttagtgccttttggtgtttgacactgacttgacaaatggaaagcaaatatgcacggtgccttttgGCATTTGAGCTGAATGGAGAGAGCTATGGACGATGGGGTTGGGTTGAACTTGCTACATGTCCGAGCCATTTGTCTTTGAAAGCTTTCTAGCGTGTTGAATATTTAACTATGGTCATTCCTTCTACCTCACCAAAACAGTTATGAATTCTGTAAAACCACTAGATGCTTGTTGACAT
This mutation is a 2177 bp deletion beginning at Chromosome 10 position 85,927,535 bp and ending after 85,929,711 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
44815 | AGG AAA ATA CTT CGT GCT ATG G | Common | A | |||
44816 | GTT ATT TCT CGG AGT GCT TAC TGT | Wild type Reverse | A | |||
44817 | AAG TTC AAC CCA ACC CCA TC | Mutant Reverse | A | |||
44818 | Fluorophore-1 | AGA GGT TGG GAG GCA GGA | Quencher-1 | WT Probe | ||
44819 | Fluorophore-2 | CTG AGT GGC ATT TGA GCT GA | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
44815 | 0.40 uM |
44816 | 0.40 uM |
44817 | 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 |