Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr9:104082614+104082708 95bp TCTAACTCAGAGCCATACAGTGTC CATATGGGGAACTAAATTCAC
Mut= 80 bp
Wt= 95 bp
Fam=Mut
Hex=Wt
Wt Sequence (deletion in lower case):
TGAAGAGAAACAGTTATCAGTACAGATGCTGTGACCTGCAGGTGGCCTTGTTTGGGGCAGTGGCTCATTTTCAAGTTTGGTTTGAGTCATTGGTTATACTTCTGCCTGGATGAGAGTGTCACAACTATGTGATATGAGTGTGACTCTAACTCAGAGCCATACAGTGTCCTCTGCAAGTATGTTCCTGGTCTCcatggtctgactgacgtctggagtccgtgaatttagttccccatatggatcacacactggaaagactaattgggtagccggggatcgttttcaatgaactttaattgtcccatgttaatttacttgtagggagtctatcgtagatacctcatgggaaataactcatctgaggatagcttcttaactgccaatactgtgcaacctctggcagaaactggattgcagctgtcaaaaaggtaagggaagcatattctctgggtaattcaactggcttggtctcctaagaccgcagagtcaggagacagtctgggttcgtccagttaaatagtcactggatgtgagaaggttaaaatgttaagaaaatggaaatgatatctctgcccagacTAGGGCTGCTTTGCCAGTCAGGATAGTTTTCTAGTAAGTCACTATATACCTAAAAAGATATTGACTTAAAAATGAAAAAAAAATTGCCCATATTTATGATTAAGTGTATTCTTTGCCACTTTGAATGTATAACCTTTTAAATACATAGGTGAATATAGTTTCTTATATTTGTTACACATAC
This mutation is a 387 bp deletion beginning at Chromosome 9 position 104,082,662 bp and ending after 104,083,048 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
46223 | TCT AAC TCA GAG CCA TAC AGT GTC | Common | A | |||
46224 | CAT ATG GGG AAC TAA ATT CAC | Wild type Reverse | A | |||
46225 | AGA AAA CTA TCC TGA CTG GCA AAG | Mutant Reverse | A | |||
46226 | Fluorophore-1 | ATG GTC TGA CTG ACG TCT GGA | Quencher-1 | WT Probe | ||
46227 | Fluorophore-2 | TTC CTG GTC TCT AGG GCT G | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
46223 | 0.40 uM |
46224 | 0.40 uM |
46225 | 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 |