Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr5:138151258-138151353 96bp GTGACCATGGGTCCGAAAG CCTTGTCAGGAAAGGAAGG
Mut= 96 bp
Wt= 96 bp
Fam=Mut
Hex=Wt
Wt Sequence (deletion in lower case):
TTGGAGTTCAGTGGTAGAACACTTGCTTGCATGCACAAAGCTGTGGGTTTGATCCCTAGACCCACCAAGAATTTAAAAAAAGAAAACAATATGGCACTCATGAAAGGAAAGACTGGGTGGTTTGGTGGATTTTTGAGACATGTGACCATGGGTCCGAAAGCAGGTAGATTGATTCctagccctggtattgtccttcagctctttcttcttcaaaagtcccttcctttcctgacaaggacagtctgggggacaagatgttggctgctgcgcttctaaaggccaagtctcaggtaagtcatggttcccttcattcttttccttttccttttgatgtctcattcagtgccctaaattctaattcTGTCCATCTAGACTACAGATCTGGGGGCACTGAGATGATGTAGTGCATAAAGGTGTCTGCTGCCATCCTGCGAATCGGAGTTCAATCCCTAGTAGCCACATGTTAGAGAAAACTGGCTCTCAGATTGGCCTGTAACCTCCATGTGTGTACAGTAATAGCCCTTCCCCAAATAAATACATGTTAAT
This mutation is an 186 bp deletion beginning at Chromosome 5 position 138,151,134 bp and ending after 138,151,319 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
44724 | GTG ACC ATG GGT CCG AAA G | Common | A | |||
44725 | CCT TGT CAG GAA AGG AAG G | Wild type Reverse | A | |||
44726 | CAG CAG ACA CCT TTA TGC ACT AC | Mutant Reverse | A | |||
44727 | Fluorophore-1 | AGC CCT GGT ATT GTC CTT CAG | Quencher-1 | WT Probe | ||
44728 | Fluorophore-2 | AGA CTA CAG ATC TGG GGG CAC | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
44724 | 0.40 uM |
44725 | 0.40 uM |
44726 | 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 |