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Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr3:96893600-96893722 123bp GCCTAGTTGGCTGATTCCA GGGAGCTGAAATTAACAGCAA
Mutant= 115 bp
Wild Type = 123 bp
The common forward primer (primer 65572) anneals over the nucleotide sequence containing mouse genomic variation rs37776457.
Wt Sequence: gcctagttggctgattccatgggtcgtattcaGcataggctccaagaactagagaccttaggaatactaaaagcatgagagtcgccgcttccctgcaaacctttgctgttaatttcagCTCCC
Mut Sequence: gcctagttggctgattccatgggtcgtattcaGGgcatattgaatgctggtcaactctgttattgtaacaagagttaatcggttaaaaggcagaaagatttattttagctcccag
A a 1011 bp deletion beginning at Chromosome 3 position 96,892,679 bp and ending after 96,893,689 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
65572 | GCC TAG TTG GCT GAT TCC A | Common | A | |||
65573 | GGG AGC TGA AAT TAA CAG CAA | Wild type Reverse | A | |||
65574 | CTG GGA GCT AAA ATA AAT CTT TCT G | Mutant Reverse | A | |||
65575 | Fluorophore-1 | TCG TAT TCA GGG CAT ATT GAA TG | Quencher-1 | MUT Probe | ||
65576 | Fluorophore-2 | ATA GGC TCC AAG AAC TAG AGA CCT TAG | Quencher-2 | WT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
65572 | 0.40 uM |
65573 | 0.40 uM |
65574 | 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 |