Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr8:35825399-35825526 128bp GAAGACAGCCACCTCCCAAG CAAGTCACCCTCAGACTCCAG
Mut = 134 bp
Wt = 128 bp
Fam = Mut
Hex = Wt
Wt Sequence (deletions in lower case):
GGCTGGGCGAGAGGGCGGAGCGGTTGGGACGCGAGAGAGGAGGGGACTTGGTGTGAGAGGCTCAGTGGGGACAGTTCCGCGCGAGAGACAAAGAGCGCCCAGAGAGCGCAGCGAGCATCGCTGCTACCCAGCCCGGCAACCTCGCAGATTCGAGGACAGGGTGCGCAGCGGAGCAGCCACAGCTGCTAGGGACCCAAGAGGCTACGCAGGATGCGGACGCCGGTggtgatgacgctgggcatggtgctcacgccctgcgggttgctgcttaatcttgtcagtacactggccccgggctggcggctggtgaagggctttctggaccagccagtggacgtggtgctgtaccaggggctgtgggacatatgtcgcgagcagagcagtcgcgaacgcgagtgcggccagcccgacgagtggaactacttccagacccagcctgtgcaggtggcccggggactcatgatcacgtcactggccactaccgccctagggctgctgctggcttccctcggtgtgcgctgttggcaagatgagccccactacgggctagcgggcctctctggcgtggtgtttttcgtcgctggcctcttcagcctcatcccggtctcctggtataaccacttcttgtcagatcccgacgtcctggccgccccgagctcgccggtcacggtgcaggtcagctacagcctggtgctgggctacctaggcagctgcctgctgcttctgggcggcttctctctggcgctcagctttgcgccctggtgtgaagagcgttgtcgccgctgtcgcaaggcgcccccagcaggcccgcgccgcagcagcatcagcaccgtctacgtggactggccggaaccagcgctcacacctgccatcaagtactacagcgacggacagcatcggcctcctcccaccgcagagcacagggacaccagcaagctcaaggttggattcccgatgccacgaccgccacccaagtcctacaccaacccgatggatgtgcttgagggagaagaaaagaagacagccacctcccaaggtggttcctcctctcgcagcactcggccctgccaaaattcgctgccctgtgactccgaCCTGTAGACAGGGTTCACCTGAGCTCTGCCTGGAGTCTGAGGGTGACTTGAACTTCTGCCAGTACATCTGCAACTAGAACCCTGGTCGCCTTCCTCTAACTGGAGGGCAAGCCTTCCTTTTCTAGGGCCAAACTCCTCTCCAAATACTAGGTTGGGTTCATTTATCTTTTAAGGAGTTTTACTTTTCTCTCCAGAG
This mutation is a 860 bp deletion beginning at Chromosome 8 position 35,825,454 bp and ending after 35,826,313 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
49325 | CAA GTC ACC CTC AGA CTC CAG | Common | A | |||
49326 | GAA GAC AGC CAC CTC CCA AG | Wild type Forward | A | |||
49327 | CTC GCA GAT TCG AGG ACA G | Mutant Forward | A | |||
49328 | Fluorophore-1 | CTC GCA GCA CTC GGC C | Quencher-1 | WT Probe | ||
50972 | Fluorophore-2 | AGG CTA CGC AGG ATG CGG | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
49325 | 0.40 uM |
49326 | 0.40 uM |
49327 | 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 |