Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr3:137864556+137864710 155bp AGTATAAAGGGCGCGAGGA ACTCTTCACGTTACCATCTCG
Mut= 162 bp
Wt= 155 bp
Fam=Mut
Hex=Wt
Wt Sequence (deletion in lower case):
GGACGCCAGCGCGCGCTCCTCCCCAGCCTTCCAGCTCCTCCCTCCAGGCACGTGACACCACCGTCCACTCGCTCGGTGGCGCAGCGCTCTGTTGCTGCCCTCCCGCCGCGGCGCCCCTTCTTCTCCCGGTCCTCGTCCCGCCGCATCTCCTCCCCCCCTAACTCATCCCCCACGCGCCAATCATCGCTCGAGCTCCCGAGCGCCCGCCCCGCCACTCCGctgtgcgttctcccattggctggagcctcaaggacgcgtcccgagggcgggaggcaaccattggtgggccgaaccggccgagttcccggatgagggaacattctgcagtataaagggcgcgaggaaggcgggagacggcgcagtttgaatcgcggtccgacggaggagtgggcgctgggatctcgctgagcgtccgcctggcctcgtctcttcctcgctcgtcggagcttcagcacggtccgagatggtaacgtgaagagtgacggcgggcggccgcgggctgcaggctccgagcgggcggggccgccgtccccggggctgggagaggcggcggcggcggcgagtgggagcgtgcggggacggggtcgggacgagcggaggggaggtgttttcggagccccggagctccaggcgggaggcgacggttgggggggcgcgcacggcgcggctgggagccggcgcgggggcgcgcagccgcggcgggctcgcggcaggggtcggcgggggagaagggcgggcggggggcgagcggcggcccggggtccccggcggggcgggcggcgggcggccgagcgcgcgctcgctcgcctccaggacgcgctgtggtgggggaggggcgcgccgccttggtaattctatcttctccgtccctcccgggctcgcgggcgtcggcggcgccgcaggctggcggtaaggctggaaaggactccggaaaggccaagacaaaggcggtttcccgctcgcagcgagccggcttgcaggtgggtagcaactgccgcagtcgggtgaccgggcctttatgtttgcaccccccttgcttgtttgtctgcaccctccatgggtgttccatcttagcatgagtgatgtgtcgcgacttggccctgcggagacagtgaatactggggtttggggagggggggcgtcggatcacatgtgtgttaactggcggggcgcgttggtgcaccaccaccgtccttggctttggcgcgcacatcttatcttaacgaatttcctatctttgtgcattttttttcttgtttgttcgtttgtttgtgtgcataaaaatcaagttccctgtgggccgtattcatcgacacctgaaatctaggacaaccagccacggacgtgtgggcgcgaccgccgctgtgtacagcgcagccatcctggagtacctcaccgcagaggtaagcgggagtacgcctgtgatctggaaaagctctgggggggcgggggaaagtggcgagagaggaggaaagtgatgcaagaaaactcgcaggccctaagagcggcgcgcagaccgagggagcgctagagggagctcgcgttcagccgaggaggctgctgggccagagtgtgctgctcctgcaaatgcaattgcgtgccccttcggggttttttgggtggttttttttttttttttgcataattctgcataattttatcattcaggtagtagggaatgacaacagttggtaaataacaccattgaggttcaaagatcttgattgaaagtaagttggaatattgatcatcagatttttatttattttttgccttttaggtacttgagttggcaggaaatgcgtcaaaagacttaaaggtaaagcgtatcacccctcgtcacttgcagcttgctatacgtggagatgaagaattggattctctgatcaaagctaccattgctggtggtggtatgtcatccctaaactcctaacattctatttaagaagaaaattcctacactcccattgtttctagaagagatttcagtacagggtgctttcatggggtttggctttccacttagtttttgctaagatcattgttcatccctcaagttttcactttgtatatgaattttacctgctttgagttcatgtattgtttttttaaatgtttatccagaaggtagacttaatctgtgtccctctcctaggtgtcatcccacacatccacaaatcgctgatcgggaagaaaggacaacagaagactgtttaaggatgcctggattccttattatctcaggactctaaatattcctaacagctgtccagtgttggtgattccagtggactgtatctctgtgaaaaacacaattttgcctttttgtaattctatttgagcaagttggaggcttaattagccttccaaccaaccaaatttctgcattcgagtcttaaccatatttaagtgttactgtggcttcaaagaagctattgattctgaagtagtgggttttgattgagttgactgtttttaaaaaactgtttggattttaattgtgatgcagaagttatagtaacaagcatttggttttgtacagacattgtttccactctggtggataagctcaataaaggtcatatcccaaactagctttaaacttgcttaataatcgggacttaccttagatctcactcagcaacaagtacattctctgcttactaattaaacagtgcatctgtagtcataaccccgtggtacttttgttgtttctttgtatcatatgacctcttagggtcttaaaacttgggaagaagctggaggtaactgcttttggcgtcccccccccccccaattgacagttaactaatgtgaaagggcctgtatcctaaggtgggacacaagtacagagaccctcagggtacaacgaaagtacctccaCCGAATGAACTTCGGATATAATTTGCTTTTAGTGACCAGACACAGGTGTGAAATGCTAGTCACATCTTGATTCATGCCAAGCATGTTCCATTATTATACATTGACCCCTTTCCCCTCCCTGAGCCTTAAGACATGTATCCTAATTCCTGGAGCAAATCAAACAGAATAGGAGGTGCTTAAGCAGCCTATGTGGATGAAAGAACTTCCTGCCTTACAGCTG
This mutation is a 2664 bp deletion beginning at Chromosome 3 position 137,864,450 bp and ending after 137,867,113 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
52793 | AGT ATA AAG GGC GCG AGG A | Wild type Forward | A | |||
52794 | ACT CTT CAC GTT ACC ATC TCG | Wild type Reverse | A | |||
52795 | TCT TCT CCC GGT CCT CGT | Mutant Forward | A | |||
52796 | GAC TAG CAT TTC ACA CCT GTG T | Mutant Reverse | A | |||
52797 | Fluorophore-1 | CGC AGT TTG AAT CGC GGT C | Quencher-1 | WT Probe | ||
52798 | Fluorophore-2 | CCA CTC CGC CGA ATG AAC TT | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
52793 | 0.40 uM |
52794 | 0.40 uM |
52795 | 0.40 uM |
52796 | 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 |