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Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr5:73306840+73307003 164bp CACCTCTGCTACTGTCTGCTG CAAACTAACAAGAAGCCCAAACTG
Mut= 173 bp
Wt= 164 bp
Fam=Mut
Hex=Wt
Mut Sequence:
catgtttatgaaacctgatctgaaggaggcttttctttgtgagagacacctacccccaaagctaaatatttagaaaatggaatgtatgtttggaaactgaagttttagcttaagatgttattatgttaatatgtctaactcttcaagatatttaccaaagtaaattatttttagaagaatagtcacttaagagtgggtgagatgactcagcaggtagagccact
This mutation is a 4204 bp deletion beginning at Chromosome 5 position 73,306,259 bp and ending after 73,310,462 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
52757 | CAC CTC TGC TAC TGT CTG CTG | Wild type Forward | A | |||
52758 | CAA ACT AAC AAG AAG CCC AAA CTG | Wild type Reverse | A | |||
52759 | GGA GGC TTT TCT TTG TGA GAG | Mutant Forward | A | |||
52760 | CCC ACT CTT AAG TGA CTA TTC TTC | Mutant Reverse | A | |||
52761 | Fluorophore-1 | AAA GAT GGG CAA GGC CTG | Quencher-1 | WT Probe | ||
52762 | Fluorophore-2 | AGA CAC CTA CCC CCA AAG CTA AA | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
52757 | 0.40 uM |
52758 | 0.40 uM |
52759 | 0.40 uM |
52760 | 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 |