Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr1:170302458-170302615 158bp TTCAAGCCTGGGCAGTGT TGTCCACGTTCCTTCCTCT
Mut = 165 bp
Wt = 158 bp
Fam = Mut
Hex = Wt
Wt Sequence (deletions in lower case):
AAAAACAAAACAAAACAAAACCCTGGAAAGTTTAAAAACATCTTTACTGGAAGCTTCCAGaaggtttggggtatattgcaaaaccatcaaagctgagtgacaaatccagggtcctgttcccctccacttacatgtctctctctctctctctctctctctctctctctctctctctctctctctctctctctctctctctttcttttctttgtccatagttgaccacagtatgtttgagaatttgaacacgaccctcactccaaagctccagtccagccattcctttccccacctgtccagacctggggccccaggctctatcgcccctggttctggagagccaggaggaccaggactgagggtgggcagcagccagcaccttaggaatctgggcaaagctgtgggggccaaggtcaatgacctcctgcggagaaaggagtcttcaagcctgggcagtgtgggtgtattggagattaataagacggctggagcccagctgcctggtggggaagacgcagcctgtggaccctggctggaggatgaaaggtgagtcctctcaaccccccattatcccagTTGTAGAGGAAGGAACGTGGACAAAACCACAGAGCATACAGGAAAGATCAGACCCA
This mutation is a 516 bp deletion beginning at Chromosome 1 position 170,302,481 bp and ending after 170,302,996 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
53140 | TGT CCA CGT TCC TTC CTC T | Common | A | |||
53141 | TTC AAG CCT GGG CAG TGT | Wild type Forward | A | |||
53142 | GGG GAA CAA CTG AAA TGT TTG | Mutant Forward | A | |||
53143 | Fluorophore-1 | CTG GAG GAT GAA AGG TGA GTC C | Quencher-1 | WT Probe | ||
53144 | Fluorophore-2 | CAT CTT TAC TGG AAG CTT CCA GTT G | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
53140 | 0.40 uM |
53141 | 0.40 uM |
53142 | 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 |