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Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
Mut = 100 bp
Wt = 90 bp
Fam = Mut
Hex = Wt
>chr3:104055178-104055267 90bp AGTAAACCCCGCTCCTCTTG CTTTCCATTAGGTCAAAAGGTTAGG
MUT Sequence (junction in uppercase):
gcttcagcacctttgacctctggtcctcctctggttttcctgtgctctctctcagccctttTTtttctcacaccctgaaagagctgcctcctttcttctggagagcagctccttgctatgaaaatg
This mutation is a 7616 bp deletion beginning at Chromosome 3 position 103,956,433 bp and ending after 103,964,048 bp (GRCm39/mm39).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
57961 | AGT AAA CCC CGC TCC TCT TG | Wild type Forward | A | |||
57962 | CTT TCC ATT AGG TCA AAA GGT TAG G | Wild type Reverse | A | |||
57963 | GCT TCA GCA CCT TTG ACC TC | Mutant Forward | A | |||
57964 | CAG AAG AAA GGA GGC AGC TC | Mutant Reverse | A | |||
57965 | Fluorophore-1 | CTC GTA ACC ACC GAG CCA TCT | Quencher-1 | WT Probe | ||
57966 | Fluorophore-2 | CTG TGC TCT CTC TCA GCC CTT TTT | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
57961 | 0.40 uM |
57962 | 0.40 uM |
57963 | 0.40 uM |
57964 | 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 |