Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr3:101897831-101897957 127bp GGAGAAAAGTCTATCTCACAGGTAATC CGAACTCACAGAGGACTTTCAG
Mut = 118 bp
Wt = 127 bp
Fam = Mut
Hex = Wt
Wt Sequence (deletions in lower case):
TGTTCCGTTCTGTTAGAAGTATATGAAGAATAATCTGGAAATAAACCACTCCAGCAATGGCTTCCAGCATTTCTGAAGCTTGGatatggtctaagatgaatattataccagattctggctaatagcggttgatgcagtaaggtgtcccagtctgggctgtatacaagactaatttagttcactgcatatattgctgtctgtggcctaaattgtcccctttctgtttcttcctttctttttcttgtagtggtttttaattgccaaccgatcttacaaagtcagcgcggccagctcctctttcttcagtggtgtgtttgttggtgttatctcctttggtcagctttcagatcgatttggaaggagaaaagtctatctcacaggtaatctgtcgccatatgcatacactgaataagaggctttattttcccaggactgttatggaaatactaaggggGGGTTATTTTCTGAAAGTCCTCTGTGAGTTCGCAGGTGGACAGAGAG
This mutation is a 371 bp deletion beginning at Chromosome 3 position 101,897,863 bp and ending after 101,898,233 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
52162 | CGA ACT CAC AGA GGA CTT TCA G | Common | A | |||
52163 | GGA GAA AAG TCT ATC TCA CAG GTA ATC | Wild type Forward | A | |||
52164 | CTG TGT TCC GTT CTG TTA GAA GT | Mutant Forward | A | |||
52165 | Fluorophore-1 | CCA TAT GCA TAC ACT GAA TAA GAG GCT | Quencher-1 | WT Probe | ||
52166 | Fluorophore-2 | TCC AGC ATT TCT GAA GCT TGG | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
52162 | 0.40 uM |
52163 | 0.40 uM |
52164 | 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 |