Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
chr3:158041717+158041823 107bp CTTGAACTACAGTGTGGCCCAGTA TGGGATGATGTAAAGATCTGGTC
Mutant= 110 bp
Wild Type = 107 bp
Fam=Mut
Hex=Wt
Wt Sequence (deletions in lower case):
ATCTGTTCTGTGTGTGATATTAAGAGACCACCAGAACTCTGCTGCTGTTGACGCCTCCTCAAGTCAAGTCTGTGTTTGTTTCTTTTCATTTTAGTAATGACTTATTTGTTCACAATTTCTGGACAGCAgtttggtcaatatacacagaataaattactaatcacccaacacattgccatttaatatgaaaaacaggagtttagtaaattattttttttcaatggcaaagctcaactattgctaattgtaagactaagaatattgactttgtcttgtaatcattttagatacatgataatcagctgacatctcttccttcagctataagagagctagacaatcttcagaaacttaatgtcaggtaaagtgtgaacctgtttctaaaactgaaatcccttttcttcacatagaaaataaccttgaactacagtgtggcccagtaggaatcaggcttggttaaacacctgccagatgctgtattctaccCAAGAGGAGAAAACAAGACCAGATCTTTACATCATCCCAGAGAATTTAATGAATCAATTATTTGTTTTC
This mutation is a 358 bp deletion beginning at Chromosome 3 position 158,041,427 bp and ending after 158,041,784 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
66656 | TGG GAT GAT GTA AAG ATC TGG TC | Common | A | |||
66657 | CTT GAA CTA CAG TGT GGC CCA GTA | Wild type Forward | A | |||
66658 | CTC AAG TCA AGT CTG TGT TTG TTT C | Mutant Forward | A | |||
66659 | Fluorophore-1 | ACA ATT TCT GGA CAG CAC AAG AGG AG | Quencher-1 | MUT Probe | ||
66660 | Fluorophore-2 | TCA GGC TTG GTT AAA CAC CTG | Quencher-2 | WT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
66656 | 0.40 uM |
66657 | 0.40 uM |
66658 | 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 |