Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
Mutant= 142 bp
Wild Type = 148 bp
>chr19:6939718-6939865 148bp AGGTGTGGCCATCTGAGTTC CAGTGAAGCACCCACGAAG
Wt Sequence (deletion in lower case)
CATCTGCAGAAAGAGATGAGCAGAGTTTAGGTGTGGCCATCTGAGTTCTAACTGACCCTGGGGTCTCCTGAGgagtcagggttgctggtgagggccgaggggatccgtggtctggggctgactttgctgtgggctgcaggctggctgtccgaggggccttcgtgggtgcttcactgctctttttgctggtgaatgtactgtgtgcagtgctgtctcgccagcgccaggcacagccctgggtccttctgctggtacgtgtcctggtgagcgactctctcttcgtcatctgtgccctctcgcttgctgcctgcctctgccttgtggcccgtcgagccccttccaccagcatctacttagaggccaaggtagagccaccaaccttgatgtagcacccttgggggtccttggacagagttctttaggatgtaaaggaatataggagccctcctctccatgaagatcctctattgtcatctctgccaggggaccagcgtgtgtcaggcagctgctatagggggtgccatggtccttctctatgccagccgggcctgttacaacctggcagctctggccttggcccctcggagccggctagatgccttcgattacgactggtacaacgtatctgaccaggtaggcattggtcctgtttgtcccttctttggtaactataataactagcctccagctggccagggccctgccagcagcctctgtggtggcagaatgtcctcagcccctgcttttccccaggcagatctggtgaatgacctagggaacaaaggctacctggtgtttggtctcatcctcttcgtttgggaactgctgcccaccacattgctggtgggcttcttccgggtacaccggcccccacaggatctggtagggacccccccccaaccacctcttggtcCAGTATAGAGGGCTGGACCCTGGGAATTCTGGTCTGGGGTCCTGGGACAGTTACGGCAGCAGAAGAAAATATCCATAGTACCGACTTCTTTCCCCATACCTTAC
841 bp deletion beginning at Chromosome 19 negative strand position 6,939,821 bp and ending after 6,938,981 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
37266 | AGG TGT GGC CAT CTG AGT TC | Common | A | |||
37267 | TAT GGG GAA AGA AGT CGG TAC T | Mutant Reverse | A | |||
37268 | CAG TGA AGC ACC CAC GAA G | Wild type Reverse | A | |||
37269 | Fluorophore-1 | TCC TGG GAC AGT TAC GGC | Quencher-1 | MUT Probe | ||
37270 | Fluorophore-2 | CTG ACT TTG CTG TGG GCT G | Quencher-2 | WT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
37266 | 0.40 uM |
37267 | 0.40 uM |
37268 | 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 |