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Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr16:32950183+32950302 120bp CAGTTTCCACTAGGAATTCTGGA TCGTGACAGGTCTGAAAGAAAA
Mutant= 80 bp
Wild Type = 120 bp
Wt Sequence: cagtttccactaggaattctggagaaatcaagtaattatgaagcatactaActaatttcagtaaaacaagtttcaaaggatggttttaagcttttatattttctttcagACCTGTCACGA
Mut Sequence: cagtttccactaggaattctggagaaatcaagtaattatgaagcatactaAGcctggtatgcacaagacactggttcagc
A 476 bp deletion beginning at Chromosome 16 position 32,950,234 bp and ending after 32,950,709 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
65582 | CAG TTT CCA CTA GGA ATT CTG GA | Common | A | |||
65583 | TCG TGA CAG GTC TGA AAG AAA A | Wild type Reverse | A | |||
65584 | Fluorophore-1 | AGT TTC AAA GGA TGG TTT TAA GCT T | Quencher-1 | WT Probe | ||
65932 | GCT GAA CCA GTG TCT TGT GC | Mutant Reverse | A | |||
65933 | Fluorophore-2 | AGT AAT TAT GAA GCA TAC TAA GCC TGG T | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
65582 | 0.40 uM |
65583 | 0.40 uM |
65932 | 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 |