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Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr19:58527763+58527865 103bp TGAGCAGTGTCAGATTCTTCAG CACCATGCTGTCTTCTGGTC
Mut= 98 bp
Wt= 103 bp
Fam=Mut
Hex=Wt
Mut Sequence:
tggtcacagcgattgattctttctaagttgtgagactagaagcctgctccttgaaacaagctgtttgatgtcttatgagaccagaagacagcatggtg
This mutation is a 2258 bp deletion beginning at Chromosome 19 position 58,525,572 bp and ending after 58,527,829 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
45078 | TGA GCA GTG TCA GAT TCT TCA G | Wild type Forward | A | |||
45079 | CAC CAT GCT GTC TTC TGG TC | Common | A | |||
45080 | TGG TCA CAG CGA TTG ATT CT | Mutant Forward | A | |||
45081 | Fluorophore-1 | ACA CTG TGA ACT GAG CAT TTG ATT | Quencher-1 | WT Probe | ||
45082 | Fluorophore-2 | AAG CCT GCT CCT TGA AAC AA | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
45078 | 0.40 uM |
45079 | 0.40 uM |
45080 | 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 |