Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
Mutant= 120 bp
Wild Type = 128 bp
>chr17:80274675-80274802 128bp TCCCAAGTAACACTTTCCTTTC TCCTCATCTAGCCTCAAAGTG
Wt Sequence (deletions in lower case):
TGTCATTTCCCAAGACAGAAACTAACAGTTCACTATAAGATGAAACCAGCAACTgaagggcagggtcccaagtcgttggctctttgattaaatgcttacacccttgcttaatttttctctgaaataagtagacattaacatccaatattttaaaatgtccccttccctagaagaggcacctctggggaggaggaagacagcgagccccagtgtggcgaggagcagggctggccagctggacaagagcctatctttcttcctgactgcagtccttgggaatatataggcccagaagaagtggagcctcctgtgccagaatgtgcagtctcacccttggcagtgcagaaactttccaggtgatcactttatcacttacagaagtctcttctctgtggatgccctatcatgacagtgcccgtagggcccaatatgctgacaactcgatcttttaaaatggagggattattattatctgggatgctttttccaaatacagaatgtagctgtccacagttgtaactccatatttgtatttaagtgactctcacgtcagtgatgtgaagacttttacacagcatctttgtcatgttttgccctcggcaggtacggcttccacactgagcactgtcagctggccctgaggatctgtgatggagacttaggggcggcactagagcatcttctccgccagtgtttctcagagacgtttggagagaggatggcgctctctgaggcagctgtttatgtgagcttgaatgagtgtgtggagcagagacaggaagagacgcttgctctcaagtccatctgcggagaaaaattcatagaaagaattcagaacagagtctggaccattggattggaactggattatttgacaaacaaattctgcaagtctaagcaaaaggaaagtagcaaaaatgtacgggacacttcgcctgaaacctgtaaattttacctcaaaggaaattgtaaatttggatcaaaatgcaaattcaaacatgaagtgcccccacatcaaatgattgggagagcagaaagaaacgtaaatgaccctcaccttgacgctgatgacgacacaacctttatgtatgagcttcagattcgcttttctaaagaccacaaatacccctaccaagctcccctggtggcattttattccaccaatgagaacctacctctggcttgtcgtttacatatttctgagttcctttatggcaaggccttggaatttgcaaaaacttcagaacctgttgtctattctctgatcacccttttagaggaagagtcagagattgtcaagttactaacacacactcaacataagtatagcgttcctcctgtgaacgtccctccagtaccttccgagaccagaataagtaaacctgcctaccgcaaaccagtggtcccaagtaacactttcctttctaatcaaatgctagaaggtacgtacagtattgctcctaggGACAGGATGGGATCAGCAGTCAGAGTAGTGAGTTTAAAAGTAAAACACTTTGAGGCTAGATGAGGA
This mutation is a 1411 bp deletion beginning at Chromosome 17 position 80,274,741 bp and ending after 80,276,151 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
66027 | TCC TCA TCT AGC CTC AAA GTG | Common | A | |||
66028 | TCC CAA GTA ACA CTT TCC TTT C | Wild type Forward | A | |||
66029 | TGT CAT TTC CCA AGA CAG AAA C | Mutant Forward | A | |||
66030 | Fluorophore-1 | ATG CTA GAA GGT ACG TAC AGT ATT GCT | Quencher-1 | WT Probe | ||
66031 | Fluorophore-2 | AAC CAG CAA CTG ACA GGA TGG | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
66027 | 0.40 uM |
66028 | 0.40 uM |
66029 | 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 |