Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr16:11128273-11128378 106bp CCTTGTCAGGTGCAAAGC CAAGCACACATCTCCTGAAGC
Mut= 114 bp
Wt= 106 bp
Fam=Mut
Hex=Wt
Wt Sequence (deletion in lower case):
AACTCCCCTTAATAAATTATGTTTTCTAAAAGTTGAGTCACCTGTAAATGTCTCCTATAGGAACTGTACTCATGGTGCTGTATACGGTTTCCTCAGACTTGTGAGCAATAGAGCAGTAGCAGCAGTGCCTGGAGATCAGTGGCTTTTGTTGACTGTCGGGCTACAATGTTGAAATTTTGGGTTGTGAGCGGTGAGACcgtggagcgtgtcattatttactgcttccctaacccacacctgcagggacaatgcccagcatctgtactgtcccagccaggtgttgccttagcatggtgtattggtttctgttcagggcggtgacttgcaagctcttaatttcttactcaaaagaggcctttaaaaaaagtcttctctcttcttccgtacaaaagtgtaatggaagaaaataagtaagtggagaaattgtttgtgtttactcttttttagtcgagcaaaagacgtgataataccagcaaagccgcctgtcagctttttctcctcgagatctccggtccttgacctcttccaggggcagctggactatgctgaccatgtccgccaggattctgaggtggtggtgctgttcttctatgccccgtggtgtggccagtccatcgctgccagggcagaaattgagcaggcagccagccggctttctgatcaggtaatgtgtgctttcacccaagcggtgactgtcaagtctatattagagaagatggcctctcagagaaatgatgccagactggccgtgatatccaggcaaactccggggccttgtcaggtgcaaagcttgtgaagaaacaggaacaagaatcctggcatgaggtcAAGTGGCAAGAGTAAGCTTCAGTTCTGTAGCTTCAGGAGATGTGTGCTTGCCCAGGATACCAGGTTGTGGGATACAGTGCTGAAGATAGTAGAGACCAAGAACCCCAGCTCTCTCATGTCCAGCTTATGTGGCTTGTAAGCCTTTTATGTTGTAAGTGGCACACAGGCCATGGTGAATGTG
This mutation is a 631 bp deletion beginning at Chromosome 16 position 11,128,323 bp and ending after 11,128,953 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
46870 | CCT TGT CAG GTG CAA AGC | Wild type Forward | A | |||
46871 | CAA GCA CAC ATC TCC TGA AGC | Common | A | |||
46872 | GAT CAG TGG CTT TTG TTG ACT G | Mutant Forward | A | |||
46873 | Fluorophore-1 | ACA AGA ATC CTG GCA TGA GGT | Quencher-1 | WT Probe | ||
46874 | Fluorophore-2 | TGA GCG GTG AGA CAA GTG G | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
46870 | 0.40 uM |
46871 | 0.40 uM |
46872 | 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 |