Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr4:97574907-97575029 123bp TTTGGAGGGGATGTAAGACG CAGGGTGTCAATCACCTACG
Mut= 118 bp
Wt= 123 bp
Fam=Mut
Hex=Wt
Wt Sequence (deletion in lower case):
AGTGATCTTGCAAACAGGAAGCGATTGAACTCACTGTGTTACATAGGAAGAGGTGAGCCATTTTTGTTGAATTTGGTTTCCTCTGAAGACTGAGCATTTTAAGAGAAGTTTGGAGGGGATGTAAGACGGCTGATTGTATGATaacgcaatggaaatgccactcttggaagttgagatcttaagtaattaatgtcataaatgtgtgtttaatcgtaggtgattgacaccctgccaggtgctcaggattccaaggggaaagacagtggcttgccctccaggtgcacactagctctcgctgaggaagcagggacaggagcaggcatgtactttgcatttggtaggaccagcgaatgtacatttgactctgtgttgtcacatgtcctttgtagtggcttcattagtaaaatagggatggtgttggccaacttacagggagtgctgtgaagattaactgaacctactattcaaaagaacaagaatactgggtacacacttagtaggcactcagcctggtttctgtttcctttatttattcttgggatcctagagctccttgcgtatctccttTGTAAATAAGAGGCTGTCATGTGATTTCTTTGGTGGCGAGGAGAATGGTCCTGTGGTATGTACTCCATTCAGATACCTCTCCAGGAAGACAGTAGCCAATATCTTGCAGCTGAACCTTCCTTCTATGGAAGAGAAGAGTTGTTTGTGCTTA
This mutation is a 425 bp deletion beginning at Chromosome 4 position 97,574,571 bp and ending after 97,574,995 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
44476 | TTT GGA GGG GAT GTA AGA CG | Common | A | |||
44477 | CAG GGT GTC AAT CAC CTA CG | Wild type Reverse | A | |||
44478 | CTG GAG AGG TAT CTG AAT GGA G | Mutant Reverse | A | |||
44479 | Fluorophore-1 | ACG CAA TGG AAA TGC CAC | Quencher-1 | WT Probe | ||
44480 | Fluorophore-2 | CTT TGG TGG CGA GGA GAA T | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
44476 | 0.40 uM |
44477 | 0.40 uM |
44478 | 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 |