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Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr6:90412353-90412458 106bp CACAGTTCCGGGTCCTCTAA CTTTCCACAGGAAGGCTGTC
Mutant= 86 bp
Wild Type = 106 bp
Wt Sequence: cacagttccgggtcctctaaaatcaagatccaGcagtggtcatatgggttctctttgcttccagcagGATCTGGCAGTAAGAGCAAGACAGCCTTCCTGTGGAAAG
Mut Sequence: cacagttccgggtcctctaaaatcaagatccaGGggagggtgaggagagggaagacagctgaggccattccacggagctacctcag
A 349 bp deletion beginning at Chromosome 6 position 90,412,077 bp and ending after 90,412,425 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
66149 | CAC AGT TCC GGG TCC TCT AA | Common | A | |||
66150 | CTT TCC ACA GGA AGG CTG TC | Wild type Reverse | A | |||
66151 | CTG AGG TAG CTC CGT GGA AT | Mutant Reverse | A | |||
66152 | Fluorophore-1 | TGA GGA GAG GGA AGA CAG CT | Quencher-1 | MUT Probe | ||
66153 | Fluorophore-2 | AGT GGT CAT ATG GGT TCT CTT TG | Quencher-2 | WT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
66149 | 0.40 uM |
66150 | 0.40 uM |
66151 | 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 |