Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr4:140831117-140831207 91bp CCATTGTCCCAACCTAGAACC TGATCAGGGTCTGTCATCCTC
Mut= 87 bp
Wt= 91 bp
Fam=Mut
Hex=Wt
Wt Sequence (deletion in lower case):
GCAGGACCTGAGCCAACCTTCTCTGGAGAGAATCCTGTCACCTGGGGCAAGTGGGGGAGGGGTGGGAGCCCCTGGACTTTGGCAAAAGTCCTGGCGCTCACTGGTTGTGGGACCAGACTGGTCATTCCCTCTCTACAGAGGATGAATAACGTCAAGGAAGGTCAAGCTGTCTAGGTCAGTAGGACAGACAGTAGCCCCAgtttgtctatctctctcgtatcctcagaaggtcctggagtcgccatacccaactttgtagccctattttctccttaaactgtgaggggctctggaacagtaccccaggtcacaattgccaaatagcttcaccttgccttctctcagggttgactctcttcccccaggtgaaggtgtcatactttgagtcacaggaggctgctgccctggcccacagtgtgctctacctaactgctgttggtgagtcccactccggcctgcctctggacacggtctggacatggatgtagtgacggtgactgaagctttcggggccttaggggggagcctcagctacccacctcacccctccgcttcttgggaacttcaaacatctccaggaccatagggattgagacccagccatgctcagagccaatagagcctagaaatgtcactgctatgagcacaatccacagtggcttcctcatgggagtcccattgtcccaacctagaaccatggagagtcataggcctctttcacccagagcccaCTGCAGCATCCTCTGAGGATGACAGACCCTGATCATAAGTCTCCAGGGGCTACATATAGTTAAGGGTACCCTGCATGTGACATAGAAACCCGGGGTCTCTAAGACATGGTCACTCAGTGCACATGGACAAGACAGGGACTTGTGTCCTTCCGACACCAGAATTGCCAATGTCGCTCTAGCCAGAGCCCCGAGGCCGACACACA
This mutation is a 532 bp deletion beginning at Chromosome 4 position 140,831,152 bp and ending after 140,831,683 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
47580 | CCA TTG TCC CAA CCT AGA ACC | Wild type Forward | A | |||
47581 | TGA TCA GGG TCT GTC ATC CTC | Common | A | |||
47582 | AAC GTC AAG GAA GGT CAA GC | Mutant Forward | A | |||
47583 | Fluorophore-1 | AGT CAT AGG CCT CTT TCA CCC | Quencher-1 | WT Probe | ||
47584 | Fluorophore-2 | AGA CAG TAG CCC CAC TGC A | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
47580 | 0.40 uM |
47581 | 0.40 uM |
47582 | 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 |