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Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr12:76427586+76427680 95bp CCCCTCAAGCTCTCATTGC TCACTGCTCTGTCCCTCAAA
Mutant= 84 bp
Wild Type = 95 bp
Wt Sequence: cccctcaagctctcattgcaagccaattcCctgtgtatctcaggatacactagatacagctgaattaaaatattctttgagggacagagcagtga
Mut Sequence: cccctcaagctctcattgcaagccaattcCCtgggcttgtgcctctgtaatggcactgtgtgattctgggagttgttcatacca
A 10249 bp deletion beginning at Chromosome 12 position 76,427,616 bp and ending after 76,437,864 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
65257 | CCC CTC AAG CTC TCA TTG C | Common | A | |||
65258 | TCA CTG CTC TGT CCC TCA AA | Wild type Reverse | A | |||
65259 | TGG TAT GAA CAA CTC CCA GAA | Mutant Reverse | A | |||
65260 | Fluorophore-1 | CAA TTC CCT GGG CTT GTG | Quencher-1 | MUT Probe | ||
65261 | Fluorophore-2 | TGT GTA TCT CAG GAT ACA CTA GAT ACA GCT | Quencher-2 | WT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
65257 | 0.40 uM |
65258 | 0.40 uM |
65259 | 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 |