Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr11:62883835+62883962 128bp CAGGACAGCATTGTGGATTG TGATATTTGTAGCTTGGCACAG |
Mutant= 118 bp
Wild Type = 128 bp
Wt Sequence (deletion in lower case):
GGTGGGCACTTGAGGGTAAGCTGCTGGCTTCCTCCTCCCCTTATCTCATGGTTGTGGGTTGCTGGCTCTTGCAGTGAAGtgtaacggagctgctgtgccttgcttttaggcaccccgtggcatccttcttccatctgttcttccgggtcagtgcggttgtggtctatctcctttgcgagctgctcagtagcagctttatcgcctgcatggtgacaattatcttgctgctgtcatgcgacttttgggcagtaaaggtaggttctgtttctattttaaaactgtatttaatatgaattcaaataacgaattgaaaggaacaggacagcattgtggattgatgttagttcattcattactttagaccttctgtgtgttgaagtgccaggcacaccgttcacataaacacTGCTCAGTTGTGGTGCCCTGTGCCAAGCTACAAATATCA
This mutation is a 327 bp deletion beginning at Chromosome 11 position 62,883,597 bp and ending after 62,883,923 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
63155 | CAG GAC AGC ATT GTG GAT TG | Wild type Forward | A | |||
63156 | GGT GGG CAC TTG AGG GTA AG | Mutant Forward | A | |||
63157 | TGA TAT TTG TAG CTT GGC ACA G | Common | A | |||
63158 | Fluorophore-1 | TGT GTT GAA GTG CCA GGC AC | Quencher-1 | WT Probe | ||
63159 | Fluorophore-2 | TGC AGT GAA GTG CTC AGT TGT G | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
63155 | 0.40 uM |
63156 | 0.40 uM |
63157 | 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 |