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Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr14:8644673-8644772 100bp CGTGCACATGGTCTATGCTC ACTTGGGGCACTGGTGAAAT
Mutant= 118 bp
Wild Type = 100 bp
Wt Sequence cgtgcacatggtctatgctcacattctcttatggcatagtgaaagaGtgaataaaaatcctgtttttgattttcataaaaatttcaccagtgccccaagt
Mut Sequence: cgtgcacatggtctatgctcacattctcttatggcatagtgaaagaGTgacttaccaggttgacatgaagtaattaagataatttaattgtatttcttggatatcactgtacgtgtca
A 442 bp deletion beginning at Chromosome 14 position 8,644,284 bp and ending after 8,644,725 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
65262 | CGT GCA CAT GGT CTA TGC TC | Common | A | |||
65263 | ACT TGG GGC ACT GGT GAA AT | Wild type Reverse | A | |||
65264 | TGA CAC GTA CAG TGA TAT CCA AGA | Mutant Reverse | A | |||
65265 | Fluorophore-1 | AGT GAA TAA AAA TCC TGT TTT TGA TTT T | Quencher-1 | WT Probe | ||
65266 | Fluorophore-2 | TGA AAG AGT GAC TTA CCA GGT TGA | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
65262 | 0.40 uM |
65263 | 0.40 uM |
65264 | 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 |