Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr19:29026989-29027077 89bp TATCTCCTGCAGCCTGGTG TCCATTTCCTATTCCAACCA
Mut= 89 bp
Wt= 89 bp
Fam=Mut
Hex=Wt
Wt Sequence (deletion in lower case):
CAGACTGGCCCTTAACTAACCCCACTAATGTGTAGCCAAGGCTTTGACCTCTGGGACCTCTTGCCTGCACTTCCTATGTGCTGGGATTACAGGCATGCCATCAACCCAGACTGGAGTTCAGTTTGGTTGGAGATTCTAGTAAGATGGCAGGCAGACCGTCTTCCCTTACTTCTCTACTCAGgcttgagtagagaaccctgggagggcaatgtgctgtctatacagaaatactgtcacatgcactcagaatgtttgcccaaatacctgagtactctacagcccagtcaagtagacaataaatttgatttcagttatattacggctctggtcaacatgcctggtgtcattccttgggctcaatttgcctaacactttttctttcttgcctgcctgtcccctctctcctcctcccctcatataaaggatttccaaggacacttccacaggcagaagccctggataaagtttatcagatagacacagtgataaatctcaacgtgccctttgaggtcattaaacagcgccttactgctcgctggatacatcctgccagtggtcgagtatacaacattgaattcaaccctcccaagactgtggtaagtgactgtagcgagctgagctgacatccactctaacaggcatagctgtgaacttctcatcctatctgaccaagactattgcacgggtcctatctcctgcagcctggtggtaagagacgtccccgtgctaatagGCTTGTTAGTCATTCAGAAGTGCTATGGTTGGAATAGGAAATGGATCCAACAGCTCAGTGTGGTGTCTGCTTAGTCTCCAGATGGTGGCATAATATTTGAGGTCCATCACAACTGGAAAAAGTGTGTCACTGGGACATCTGCAACCTATGAATGATCCTTAGCCCCTTCTCCACTCTGC
This mutation is a 552 bp deletion beginning at Chromosome 19 position 29,027,034 bp and ending after 29,027,585 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
46182 | TAT CTC CTG CAG CCT GGT G | Wild type Forward | A | |||
46183 | TCC ATT TCC TAT TCC AAC CA | Common | A | |||
46184 | AGT AAG ATG GCA GGC AGA CC | Mutant Forward | A | |||
46185 | Fluorophore-1 | AGA GAC GTC CCC GTG CTA A | Quencher-1 | WT Probe | ||
46186 | Fluorophore-2 | CCC TTA CTT CTC TAC TCA GGC TTG T | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
46182 | 0.40 uM |
46183 | 0.40 uM |
46184 | 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 |