Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr2:122174482+122174582 101bp GGGAAGGAGCTATTGAGTCCTA CTCCACCCAAGTCCATCTC
Mut = 103 bp
Wt = 101 bp
Fam = Mut
Hex = Wt
Wt Sequence (deletions in lower case):
AAAAAAAAAAACCAAAAAAACAAAAACAAAAACAAACCAACAATTGAAAACAAAGCCTCTCATCCTACACCATTAAACTAGTTGGGAAAGAAGGAATGAACCTCCCAAAGGGCTTTGTGTACTAGGATTGAATGTCGATGCTAGGCAGCCATTTGTGTGaaattccccaaggtgtgtggtgtggtgtaagtccctctccccaaatgctagtctcagtagtaatcgtggtgatgattatggaggtttccaggacaacaagatacaactgcaacaaaacctttccttggagtttctaaagctacatcagtttctgcacaacaaagaaaaggacattctaaatgacctccgggatgaggggaaactcttgaatgaggagatggaggtgaatctgaaccagattcaggagcagtgtcttgttgccaaagacatgctggcgaccattcaggcacggatggaacagcagaactccttcgacttcctcacagtgagattctgaatccaattgattatggggacacttggaaggagggaagagaaactgggagtggggggtggaaggaaagcaaatcataaccaagctcaacccaacccaaccatgtcctaacctacccaacccaacccaatttaacccaacccaacccgactaactggagcgcttgtaggtccttggggagaaaaaggaagccatgtttactttatgatcagtggaaatttttactttcatcttccttcaaaacgtgataggacaatttcactaataatgctacatggatcagttctttcttacttccccatatagaccacagaagctttatttttcaaaaaatatgtctcttatagtgtttgtagagtttgggccttgcagaaattataaactgaaatatatctaaaaagctttttgtcaaacttttgctacctgaagtctcacttctagagatcagatgattggcgatcactccagcactcagaagtctagttacccttccccctcagatcgtttctgatcttttcttggcggagtatttggaagcactcacttctcaaagagttgatcttaactatgggcttcctggctcttctcttgtttcctatgagagctggaattatagaaaatcagaaggttaccatgtgagaggtcagtttatgctgggttagcctgggatgctgcttagcaaagtctgtagaggatgaaatgttgttggcgatggcctgaggaccatctgtacaggaagccagtattgatgatcaaacctggatggctttcagtataaccagagaagttcctagagatgatgaacaggcatctgacttctctatttcttcttttctcttctaggacatcacaaagctcatagagaggcaagtgtttcttcatggttctctctaccctccttcttctacccctggagaatgtctaaaactaaactgtttgtgtgtcccttacagcatggagaaaggaatgaagacaattgtacctagacagctcatcgccaaaaagctgagcctaggccgcttcaaaggtcctatccagtatatcatctggaaggaaatgcaggctattctctctccaggtatagtgggaaggagctattgagtcctatggcccctaatctatatttcatctgtggccagagttagttctgtagtttgagagAGGATGGAGATGGACTTGGGTGGAGTTTAGTCA
This mutation is a 1495 bp deletion beginning at Chromosome 2 position 122,173,063 bp and ending after 122,174,557 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
51442 | CTC CAC CCA AGT CCA TCT C | Common | A | |||
51443 | GGG AAG GAG CTA TTG AGT CCT A | Wild type Forward | A | |||
51444 | TTG GGA AAG AAG GAA TGA ACC | Mutant Forward | A | |||
51445 | Fluorophore-1 | CAT CTG TGG CCA GAG TTA GTT CTG | Quencher-1 | WT Probe | ||
51446 | Fluorophore-2 | AGG CAG CCA TTT GTG TGA GG | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
51442 | 0.40 uM |
51443 | 0.40 uM |
51444 | 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 |