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Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
Mut = 117 bp
Wt = 108 bp
Fam = Mut
Hex = Wt
>chr3:27938649-27938756 108bp GTAACCAGTGTCAAGAATGTTCCA AACTGGCAAGTGGTGCCTAC
MUT Sequence (junction, and insertion, are in uppercase):
ctacacggccatttctcattcaaaccagcacgcaaagcctagggcgagatctatgaagccaagatttaactgtcgtgacctaacagGTCaactggttttgctgtaggcaccacttgccagtttttctctcagcctagaatcttaaaactgtgtgtgggaatatagcaatggtacctattcccacctctttggtatttttctctctttttataatttttttaaattaactgacaatttcacacatatt
This mutation is a 2249 bp deletion plus a 1 base pair insertion (T) beginning at Chromosome 3 position 27,938,683 bp and ending after 27,940,931 bp (GRCm39/mm39).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
58142 | AAC TGG CAA GTG GTG CCT AC | Common | A | |||
58143 | GTA ACC AGT GTC AAG AAT GTT CCA | Wild type Forward | A | |||
58144 | CGG CCA TTT CTC ATT CAA AC | Mutant Forward | A | |||
58145 | Fluorophore-1 | TGG ATG CAT GTG TGG ACG TAT | Quencher-1 | WT Probe | ||
58146 | Fluorophore-2 | AAC TGT CGT GAC CTA ACA GGT CAA C | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
58142 | 0.40 uM |
58143 | 0.40 uM |
58144 | 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 |