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Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
Mut = 146 bp
Wt = 139 bp
Fam = Mut
Hex = Wt
>chr15:25991365+25991503 139bp CCTTTTAAGGGTTATTCGGTCA ACTAAACGGGAGACCTGCAT
MUT Sequence (junction in uppercase):
ccttttaagggttattcggtcacatttgttggcagaaaaattctgacagtaatttttatcactcagttcttgtagtggGCaagactccatgacttaaaaaattaatattcctggtctagtaacttagtatgcatcatgaacctttccagaagata
This mutation is a 5037 bp deletion beginning at Chromosome 15 position 25,991,444 bp and ending after 25,996,480 bp (GRCm39/mm39).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
56699 | GGA AAG GTT CAT GAT GCA TAC TAA G | Mutant Reverse | A | |||
59620 | CCT TTT AAG GGT TAT TCG GTC A | Common | A | |||
59621 | ACT AAA CGG GAG ACC TGC AT | Wild type Reverse | A | |||
59623 | Fluorophore-1 | ATT TGG TAG CTT GGC CAC ATT T | Quencher-1 | WT Probe | ||
59814 | Fluorophore-2 | AGT GGG CAA GAC TCC ATG ACT TAA | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
56699 | 0.40 uM |
59620 | 0.40 uM |
59621 | 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 |