Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr19:47948047+47948192 146bp TGGGGACACTGGGAAAGAC GCAATTTCACCACTTCTGACA
Mut = 144 bp
Wt = 146 bp
Fam = Mut
Hex = Wt
Wt Sequence (deletions in lower case):
CTTGTCTCTTAAGGCCTGAAGAGTGTGCACCACGGATCTCCCAGGAAGCCAGACTTGGGGACACTGGGAAAGACCCCCCAAAcaaatggccacttagtctgactcctgtgttctttctgttcagcatccgagatttactgaagttcaaagaagaggtgacgaaggagcgtgaccagctcttgtcagaagtggtgaaattgcgggagaacctagctcagaccatcgaaaagcagcaggcagcagagcacgccaaggaagaagctgaaatggccatcagtcaggtctgcccaccaccttccggttccggaggacggtctgtccttccggcctgctttcccctctagtgcctctctcagatgtaaaccgttatgtccaacccaagtgttctctgtactggctgtttctctggccgcgtctcatgagcccattttgCCCAGGGTTCTTTCTGTCCGAGAGGTCACTAAGGCAGTGTATGGCTCTGCTTAAGTTTTGGGTTCAGGTTCAGGTGGTGTCCCTGCAT
This mutation is a 350 bp deletion beginning at Chromosome 19 position 47,948,074 bp and ending after 47,948,423 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
53076 | TGG GGA CAC TGG GAA AGA C | Common | A | |||
53077 | GCA ATT TCA CCA CTT CTG ACA | Wild type Reverse | A | |||
53078 | AGG CTG AGG GAT TTC TTG CT | Mutant Reverse | A | |||
53079 | Fluorophore-1 | CTG TTC AGC ATC CGA GAT TTA CTG | Quencher-1 | WT Probe | ||
53080 | Fluorophore-2 | CAA ACC CAG GGT TCT TTC TGT C | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
53076 | 0.40 uM |
53077 | 0.40 uM |
53078 | 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 |