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Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr17:23644372-23644461 90bp GGCTCTTGGAGCTCTGGAAT CCCAGCTTCCCATTTTAGC
Mutant= 111 bp
Wild Type = 90 bp
Wt Sequence: ggctcttggagctctggaatactgtcTctatcaggaggtttggtggctgagattcagaagtttgaaaaggagctaaaatgggaagctggg
MUT Sequence: ggctcttggagctctggaatactgtcTCtaaattgcaagaagttaaagatgttggtctcaggttccagcccagaccttggactctcactggccagtctttgtcaggctcca
A 638 bp deletion beginning at Chromosome 17 position 23643797 bp and ending after 23644434 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
65577 | GGC TCT TGG AGC TCT GGA AT | Common | A | |||
65578 | CCC AGC TTC CCA TTT TAG C | Wild type Reverse | A | |||
65579 | TGG AGC CTG ACA AAG ACT GG | Mutant Reverse | A | |||
65580 | Fluorophore-1 | TGT CTC TAA ATT GCA AGA AGT TAA AGA TG | Quencher-1 | MUT Probe | ||
65581 | Fluorophore-2 | CTA TCA GGA GGT TTG GTG GC | Quencher-2 | WT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
65577 | 0.40 uM |
65578 | 0.40 uM |
65579 | 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 |