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Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr19:6389271+6389355 85bp ACCCCTACAGGAGCCATTCT GTCCACAGTCCTTGTTCAGC
Mut = 84 bp
Wt = 85 bp
Fam = Mut
Hex = Wt
MUT Sequence:
cccggtaacttacgcaagccttgaacttgtgatcctctggtcgtagcttctccagtgacagggcttctgggaacccagctagtaagccTCttctgggggctggtgaccacccttagaagatgtggtattcccacatcttagtttcatttccttttgctgtc
This mutation is a 3639 bp deletion beginning at Chromosome 19 position 6,387,911 bp and ending after 6,391,549 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
50071 | ACC CCT ACA GGA GCC ATT CT | Wild type Forward | A | |||
50072 | GTC CAC AGT CCT TGT TCA GC | Wild type Reverse | A | |||
50073 | CTC CAG TGA CAG GGC TTC TG | Mutant Forward | A | |||
50074 | TGG GAA TAC CAC ATC TTC TAA GG | Mutant Reverse | A | |||
50075 | Fluorophore-1 | AGG GTC TCC TTC ATA GTG TGA GGA G | Quencher-1 | WT Probe | ||
50076 | Fluorophore-2 | AGC TAG TAA GCC TCT TCT GGG GG | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
50071 | 0.40 uM |
50072 | 0.40 uM |
50073 | 0.40 uM |
50074 | 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 |