Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
Mutant= 109 bp
Wild Type = 118 bp
>chr14:55741367-55741484 118bp TCAGGTTTTCCCTTGGAATG GTGAATGCTGGTTCAGTTGC |
Wt Sequence (deletions in lower case):
TCAGGTTTTCCCTTGGAATGGCCTATACCTGATTTCCCTGgtcttcagctattgctatgcagcctctatactgaccacagatactgaggacttgagaagcaactgaaccagcattcacacaattcattccttcttcttcttcttcttcttcttccctgaccctgtctagaggccactacttatgctctgtgtatggggcacggctgatggtgccagctggcaaaggactcatcgtgattgtctcctcccctggggggctgcagcatatgttcaatgtcccttatggtgtgggcaaagctgcggtaaggatcaaggcacaggagttggggggtacacagcagtgttgacggttggtaatgGTCCACAGCTGTCAGAGTAGTAAGACCTTCACCTCTCCCTCCCCCCCCCCCTCTCTCTCTGTCCCATGC
This mutation is a 319 bp deletion beginning at Chromosome 14 position 55,741,126 bp and ending after 55,741,444 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
66077 | TCA GGT TTT CCC TTG GAA TG | Common | A | |||
66078 | GTG AAT GCT GGT TCA GTT GC | Wild type Reverse | A | |||
66079 | GCA TGG GAC AGA GAG AGA GG | Mutant Reverse | A | |||
66080 | Fluorophore-1 | TCT TCA GCT ATT GCT ATG CAG CC | Quencher-1 | WT Probe | ||
66081 | Fluorophore-2 | TGA TTT CCC TGG TCC ACA GCT G | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
66077 | 0.40 uM |
66078 | 0.40 uM |
66079 | 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 |