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Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr16:49069147+49069236 90bp AGCAGTTAGTTCATCTCCCAGAG GTGATTTCTAACAGAAGCCAACAG
Mutant= 123 bp
Wild Type = 90 bp
Wt Sequence: agcagttagttcatctcccagagatcaagaggcaaTacgaggtcttgatgtcagcttaacccagatctgttggcttctgttagaaatcac
Mut Sequence: agcagttagttcatctcccagagatcaagaggcaaTTcatttaaagagactccttatgaactgtattgtacaagctagttaattccttctcagggaagagcccttcagtcttgcgaatagtca
A 1220 bp deletion beginning at Chromosome 16 position 49,069,183 bp and ending after 49,070,402 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
65476 | AGC AGT TAG TTC ATC TCC CAG AG | Common | A | |||
65477 | GTG ATT TCT AAC AGA AGC CAA CAG | Wild type Reverse | A | |||
65478 | TGA CTA TTC GCA AGA CTG AAG G | Mutant Reverse | A | |||
65479 | Fluorophore-1 | AGA GGC AAT TCA TTT AAA GAG ACT CC | Quencher-1 | MUT Probe | ||
65480 | Fluorophore-2 | CGA GGT CTT GAT GTC AGC TTA AC | Quencher-2 | WT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
65476 | 0.40 uM |
65477 | 0.40 uM |
65478 | 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 |