Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr9:62427751-62427839 89bp AGACTCCCAAGCCACAGTCA GACAGGCTGGGTACAGAGGA
Mut= 82 bp
Wt= 89 bp
Fam=Mut
Hex=Wt
Wt Sequence (deletion in lower case):
CACACGTCACAGCATAGACACACTATATAAACATGCTACAGTGCACACGTGCCACACACACACTCACACAGCACACACAGCACACAAATCCCATGTACCATACAGACACTCACTGTGATCACACATACACACATATCACGTATATACCATCCCATATCCACCCctgcccctagtagagctaccaaggcacagggttagctctctcctacagtgccttgggcctaggtctgctggaccctgccctctactctcgttgcttctctgctaccctcagggggatggaaacatccggtactatgagatcagcacggagaagccctacctgagctacctcatggagttccgctccccagccccacagaaaggcctaggtaaggacctggaagctgatgcaggctgctctgtgcagagcaggagatggtgtttattgtcttgtgcatacacaggatcccctttacacaatagtcagactcccaagccacagtcagcaggcagatgggaatgggacccacacgtgccacCTTCGGCAGGCCAGCTCCTCTGTACCCAGCCTGTCACTCATGGCACCAGCTTGGCTATCTCTGTCAGCAGCAGCCTGCAGGGTATGGGCTCTGGCTTCCTCCTCAGCTGTAGATGCAGAGACTCACTCCACAGTGGGGTTCACA
This mutation is a 358 bp deletion beginning at Chromosome 9 position 62,427,786 bp and ending after 62,428,143 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
41588 | AGA CTC CCA AGC CAC AGT CA | Wild type Forward | A | |||
41589 | GAC AGG CTG GGT ACA GAG GA | Common | A | |||
41590 | GAT CAC ACA TAC ACA CAT ATC ACG | Mutant Forward | A | |||
41591 | Fluorophore-1 | AGG CAG ATG GGA ATG GG | Quencher-1 | WT Probe | ||
41592 | Fluorophore-2 | ATC CAC CCC TTC GGC A | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
41588 | 0.40 uM |
41589 | 0.40 uM |
41590 | 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 |