Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr1:54763597-54763756 160bp CCATGGTGCAGAAGTTACCTG CCAGACAGAAAGACGTGAACC
Mut= 172 bp
Wt= 160 bp
Fam=Mut
Hex=Wt
Wt Sequence (deletion in lower case):
GGATTAAAACCCAACAAGGCAATGGTAATTTCAGATTAAACAGTAGTCTGTAAGTATTCTGATTTTTTTTTAAACTGGTTTTCTAAACTAAAATTTTACTGGTCGGCTTTTTTGGTGAGGATGGTAATTGTTTTCATTTTAACACAACCCTTGTTGCTCTTACTGTGTCTGTTTTGTTGTCTAGCTGGTCTGGAATTCATTCTGTATCCCATGCTGTGTCTTAATTTGTAACTTGCTTGCCTTTTCACCCAAATGCTGGGATTCTGGGATTACACACTCAATACCAATACAACTTTTAAAAGGTGGTGCTACTAATATATTGTCAAACAGCCACCACCTTTGCTACATCTCATTCGAGTCTTAGTTATGAGTCATCTTTTTTCTTCCCATGAGATTTATATAATGTCCAGTACATATTAGGTACTCAATAAATGCTCATTGATTATTTTCAATGAGGCAGCTTCCCCCTCTCCTCCcgactcccagatcaggaaatactggtggtaaaaccagctaagaatgatctttttttgtgtggtgagttgtcgcattgtcttataaaatttaactatggtcttgctgctttatttgtcaaggtcacctggacgttgtggcgctgctcattaaccatggtgcagaagttacctgcaaggataagaagggctacaccccgctgcacgctgcagcctccaatgggcagatcagtgttgtcaaacatctcctgaacctgggggtggaggtaagctctgatggctgtatcgcGGTGGGTTCACGTCTTTCTGTCTGGGTTTTCTTCTGCTCTGAAGTGAAATCCTGTTTGCATTTTTATTCAAGTGGAGAAAAACTTATTTCCTTGGTTCATGTATTTATTAGTTTATGTGAGCTGATAGAGTGTGATAGGGACAAATAGCAGGAAACCAGAAACAGACTATAGACATAGAGAAGTATACAAATGTTTATATTTCATTCTTTTCCTTTTTTCTTTTTGGTTTTGGTTTTGGTTTTGCCTTTTGAGACAGAGTTTCTCTGTAACCCTAGTTGTCCTGGAACTTGCTCTGTAGACCAGGCTGGCCACATGCACTGTCACCTCCCAGCAAACATTTATATTTTGTGCAATTTGTAATAATCAGTCATTTTAAAAGGCCTGTCGGAAGCAGACCTTGGAAGTTGTGGCTCACAGCAGGAGGGAATAAATGGATGGCAGGGATTGAATAAGAAATGTTGATGGTGAGCTTGAACCATTAGCCAGTCCCTTCTCCTTCAG
This mutation is a 286 bp deletion beginning at Chromosome 1 position 54,763,622 bp and ending after 54,763,907 bp (GRCm38/mm10).In addition, there is a single base pair insertion (T) at the deletion site.
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
52995 | CCA TGG TGC AGA AGT TAC CTG | Wild type Forward | A | |||
52996 | CCA GAC AGA AAG ACG TGA ACC | Common | A | |||
52997 | CCA CCA CCT TTG CTA CAT CTC | Mutant Forward | A | |||
52998 | Fluorophore-1 | CTG CAC GCT GCA GCC TC | Quencher-1 | WT Probe | ||
52999 | Fluorophore-2 | CTT CCC CCT CTC CTC CTG GTG | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
52995 | 0.40 uM |
52996 | 0.40 uM |
52997 | 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 |