Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr3:29993423-29993532 110bp AACCAGGACTGTAAGGAATGTGA TCAAACTCCAGCATAACACTTTGC
Mut = 102 bp
Wt = 110 bp
Fam = Mut
Hex = Wt
Wt Sequence (deletions in lower case):
CTCCCCCTTCCCCTCCCCTTCCCCTCCCCCTCCCCTTCCCCCTCCCCCCCCTCTCTCTTTCTAAGCAAACTATGGGCTGTGTTTTGTGTAACTCCATAAGAcggctggttatggtgatgaggacttttggatcccaccttgctagctaaagatccttgcactgtttctacagaagaacggcagcaccgctgtgaggactgtgaccagctctttgaatccaaggcagagctagccgatcaccagaagttcccatgcagcacacctcactcggccttctccatggtggaggaggacttgcaacaaaacctggagagtgagagcgatctccgagagatccatggcaaccaggactgtaaggaatgtgaccgagttttccccgatctgcaaaggttagagaagacatggtgaacgtgggtgtgtgctctgcagcaaagtgttatgctggagtttgacaagtttgctcgggGGAAGACTAGAGCTTCAAAACAACTGGCCAGATGTGCTGGTGGCATGCTTGGGAATCATTGCTTCCTTAAAAAGCCTTGTCCTTAAGATACTTACAGTGGACCTGGATCATTTTTGTCAATCAGAAAATATTCCATCCCTTCC
This mutation is a 365 bp deletion beginning at Chromosome 3 position 29,993,409 bp and ending after 29,993,773 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
49716 | AAC CAG GAC TGT AAG GAA TGT GA | Wild type Forward | A | |||
49717 | Fluorophore-1 | ACA TGG TGA ACG TGG GTG TG | Quencher-1 | WT Probe | ||
51387 | TCA AAC TCC AGC ATA ACA CTT TGC | Wild type Reverse | A | |||
51388 | AAG CAA ACT ATG GGC TGT GT | Mutant Forward | A | |||
51389 | AGC AAT GAT TCC CAA GCA | Mutant Reverse | A | |||
51390 | Fluorophore-2 | AAC AAC TGG CCA GAT GTG CTG | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
49716 | 0.40 uM |
51387 | 0.40 uM |
51388 | 0.40 uM |
51389 | 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 |