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Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr8:66513417+66513525 109bp CAAACCTCTGGATGCTGTCA ACACACAGCTTCACCAATGC
Mut = 115 bp
Wt = 109 bp
Fam = Mut
Hex = Wt
MUT Sequence (junction in uppercase):
tgggacattgggacactgcaaaacaaagcggttgtgtgtgcgcacgcgcgcgcggcttaagaccaggggcgcatgcgcagactttccTGataggatgctaaataatcaacaggagtggggacaatgtacgtcgttgtcaaagttacttataaaagagctttctcaaagataaagatcaactttagcaagagtcgatagttggtttttt
This mutation is a 2265 bp deletion beginning at Chromosome 8 position 66,511,711 bp and ending after 66,513,975 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
50695 | CAA ACC TCT GGA TGC TGT CA | Wild type Forward | A | |||
50696 | ACA CAC AGC TTC ACC AAT GC | Wild type Reverse | A | |||
50697 | GGG ACA CTG CAA AAC AAA GC | Mutant Forward | A | |||
50698 | TTG TCC CCA CTC CTG TTG A | Mutant Reverse | A | |||
50699 | Fluorophore-1 | TGC CAA AGC CAC AGG TG | Quencher-1 | WT Probe | ||
50700 | Fluorophore-2 | CAT GCG CAG ACT TTC CTG ATA | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
50695 | 0.40 uM |
50696 | 0.40 uM |
50697 | 0.40 uM |
50698 | 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 |