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Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr19:57370892+57371019 128bp CAACAGACAAGAGGAGACAGAGG GTGGCTATCTCTGTTAGAAACTGTG
Mutant= 124 bp
Wild Type = 128 bp
This is a 499 bp deletion beginning at Chromosome 19 position 57,370,946 bp and ending after 57,371,444 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
47445 | CAA CAG ACA AGA GGA GAC AGA GG | Common | A | |||
47446 | GTG GCT ATC TCT GTT AGA AAC TGT G | Wild type Reverse | A | |||
47447 | TGG CTA AGT AGG AGT GTG GTC A | Mutant Reverse | A | |||
47448 | Fluorophore-1 | CTG ACG GGC TAT GTT CCA G | Quencher-1 | WT Probe | ||
47451 | Fluorophore-2 | CAG TTT CTA GCT ATG GCA TAC AGA GAG | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
47445 | 0.40 uM |
47446 | 0.40 uM |
47447 | 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 |