Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr2:172376297+172376396 100bp GGTCTCGGTGATCAGCCCTA CCATACCCTGCTCATCTGC
Mut= 113 bp
Wt= 100 bp
Fam=Mut
Hex=Wt
Wt Sequence (deletion in lower case):
TGGCAGGTGTGAGGTCCTGGGTTGTCCTCAGCAGCACAGAATAAACCAAGTGTTGTTTTCCATTCTGAATGGGAAAGATGGAATAATGGCCACACTGCTGCTTGCGTTGCACTTTGTTGTCTGGTTAGAGcaagtgaatggctgtggaggcggaaatcccctttcccagcccactctgcccatggagtgcgcctcacatagcatggaatggacagggctcacgatccatccgctccaggatgggtctttgcatttacagatcttctcatgctcttctcttgattcctttagggatggagaacgatgacactgcggtccagtatgccattggtcgctctgacacggttgccccgggcacagggatcgacctggagtttgatgcagacgttcaaacaatgtcaccagaggcttccgagtatgaaacctgttatgtcacttcccacaaaggcccgtgccgcgtggccacctacagcagagacgggcagttgatagccacaggatctgctgatgcttccataaagatactggacacagaaagaatgttggccaaaagtgccatgccaattgaggtaccattcccgcctaccactgctccagacagcgtggctgtgttgtcaccagctctggtttgccccgtgggactgtgggcgtggatccatgcctctgtgccttgagcatctatctctccttttcttgttggtccaggtcatgatgaatgagactgcacaacagaacatggagaaccacccagtgatccgaactctttacgaccacgtggatgaagtcacgtgtcttgcttttcacccaacagaacagatcctggcctcgggctcaagggattatactctgaaattgttcgattattccaaaccgtctgcaaaaagagctttcaaatacattcaggttagacctaacaggagcacttgactttctgatacagtatttcaagtggtctcggtgatcagccctacctggtacttagataagatgccattgagGGGTCACTGGCTGTCCTTTGCCCAAGCAAGACTGCAGATGAGCAGGGTATGGTAGATCACACAGTAATCCCAACACTGAGGAGGTCTGCCACAAGTTCAAGGCTAACCTGCTGCATAGTCAACTCCTTGCCAGCTTGAACCA
This mutation is an 868 bp deletion beginning at Chromosome 2 position 172,375,477 bp and ending after 172,376,344 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
45364 | GGT CTC GGT GAT CAG CCC TA | Wild type Forward | A | |||
45365 | CCA TAC CCT GCT CAT CTG C | Common | A | |||
45366 | TGG GAA AGA TGG AAT AAT GG | Mutant Forward | A | |||
45367 | Fluorophore-1 | CCT GGT ACT TAG ATA AGA TGC CAT T | Quencher-1 | WT Probe | ||
45368 | Fluorophore-2 | TTG TCT GGT TAG AGG GGT CAC T | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
45364 | 0.40 uM |
45365 | 0.40 uM |
45366 | 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 |