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Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr6:28431510+28431606 97bp AGATGCTGAGGTGTGTGCTG TGAACGAAGTTGCAAGGTAGG
Mutant= 92 bp
Wild Type = 97 bp
Wt Sequence: aaggtggaggagggagaagtacagggaaagccagcaattagcacagCcaaggggctggggcggagctaagaaggggctggggcggagctaagaagggtctggggcggagctaaaaagctgcttgcataccttctcctc
Mut Sequence: aaggtggaggagggagaagtacagggaaagccagcaattagcacagCCctagacatactttctccttccccggcatgtgtggcgagataaga
A 263 bp deletion beginning at Chromosome 6 position 28,431,420 bp and ending after 28431682 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
65461 | AAG GTG GAG GAG GGA GAA GT | Mutant Forward | A | |||
65462 | TCT TAT CTC GCC ACA CAT GC | Mutant Reverse | A | |||
65464 | Fluorophore-1 | CAA TTA GCA CAG CCC TAG ACA TAC T | Quencher-1 | MUT Probe | ||
65940 | AGA TGC TGA GGT GTG TGC TG | Wild type Forward | A | |||
65941 | TGA ACG AAG TTG CAA GGT AGG | Wild type Reverse | A | |||
65942 | Fluorophore-2 | TCT GAG CGT TTG ACC CAG AT | Quencher-2 | WT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
65461 | 0.40 uM |
65462 | 0.40 uM |
65940 | 0.40 uM |
65941 | 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 |