Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr4:88721927-88722011 85bp GCTTCTCCCATGAAGGTGTC ACCGCGTAGTTCCAGACTTG
Mut = 104 bp
Wt = 85 bp
Fam = Mut
Hex = Wt
Wt Sequence (deletions in lower case):
TGCGGCCTGCAGCTCGCAGCGAGCTCCGGCTTCTCCCATGAAGGTGTCTCTGGGtaacggcgacatgggcgtctctgcccacctgcagccttgcaagtctggaactacgcggttttttaccagcaacacgcacagttcggttgtattgcaaggctttgatcagctcagaatagagggcctactttgtgacgtgacgctggtacctggcgatggagaggaaatcttcccggttcatagagccatgatggcgtctgccagtgattatttcaaggccatgttcactggaggaatgaaagagaaagatttgatgtgcatcaagcttcatggggtgaacaaagttggtctgaagaaaatcattgattttatttatactgcaaaactgtctcttaatatggacaatcttcaggacactcttgaagctgccagctttctacagatcttgccggttttggatttttgtaaagtatttcttatatcaggcgtctctttggataactgtgtggaggttggccggattgctaacacctacaatcttatagaagtggataaatacgtaaataatttcatcctgaagaactttcctgccttgttgaatactggggagtttctgaaactcccctttgaacggcttgcctttgtgctttccagcaatagtctcaagcactgttcggaacttgagctcttcaaggcagcctgccgctggctgaggctggaagaccctcggatggactatgccgcaaaattaatgaagaatattcgctttccactgatgacaccacaggatctcatcaactatgtgcagacagtagatttcatgagaacagacaatacctgtgtgaatttacttttggaagcaagcaattaccaaatgatgccgtatatgcagccggtgatgcagtcggacagaactgccattcgatctgattctactcatttggttaccttaggaggagttttgagacaacagctagttgtcagtaaagagttacgaatgtacgacgaaagggcgcaggagtggaagtcgttagcgcccatggatgctcctcgataccagcatggcattgctgttattggaaatttcctttatgtagttggtggccagagtaattatgatacaaaaggaaaaacggctgttgatacagttttcagatttgatcctcgttataataagtggatgcaggtggcatcattaaacgaaaagcgcacattctttcacctgagtgccctcaaaggacatttatatgcagttggtgggcgcagtgcagctggtgaactggccacagtagaatgttacaacccaagaatgaatgaatggagctacgttgcaaagatgagtgaaccccactatggccatgctggaacagtatatgggggtttaatgtatatttccggaggaatcactcatgacactttccaaaatgagctcatgtgttttgacccagatacagacaaatggacacagaaggctcctatgactacagtccgaggtctgcattgcatgtgtacagttggagacaagctgtatgtcatcggtggcaaccacttcagaggaacaagtgattatgatgatgtcctaagctgcgaatactattcgccaactcttgaccagtggacaccaattgctgctatgctgagaggtcagagtgatgttggagttgcagtctttgaaaacaaaatctatgttgttggtggatattcttggaataaccggtgcatggtagaaattgtccagaaatatgacccagaaaaagacgagtggcataaagtttttgatcttcctgagtcgctgggtggcattcgggcttgcactctcacagtttttccacctgaagaaaaccctgggtcaccttctagagaatcacctctttcagcaccttcagATCATTCTTAGATCCAAGGTGTAATACCTTTTCAGTGTATCATGGGTGGTCTTGTTCATCCCCTTGTATTGTTTCTCCTTATTGCTACAGTTAATAGACAAAAAGGTAACTTAGACCCT
This mutation is a 1826 bp deletion beginning at Chromosome 4 position 88,720,160 bp and ending after 88,721,985 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
51077 | GCT TCT CCC ATG AAG GTG TC | Common | A | |||
51078 | ACC GCG TAG TTC CAG ACT TG | Wild type Reverse | A | |||
51080 | Fluorophore-1 | ATG GGC GTC TCT GCC C | Quencher-1 | WT Probe | ||
52620 | GGA GAA ACA ATA CAA GGG GAT G | Mutant Reverse | A | |||
52621 | Fluorophore-2 | CCT TTT CAG TGT ATC ATG GGT GG | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
51077 | 0.40 uM |
51078 | 0.40 uM |
52620 | 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 |