Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr12:75888482+75888621 140bp GTGGGCTTGGCTGATTCTG ACACAGTGAGACCCCCATTC
Mut= 135 bp
Wt= 140 bp
Fam=Mut
Hex=Wt
Wt Sequence (deletion in lower case):
TGCCCTGTGCCTGCCTATTCCCTGTTTCTAAATGTTCCTTTGTTCTCAGTAGGTTGTAATCGGAGGGTCTGCATTGGAGGGTCTGCATTCCAGTCTGCCTGTTCTCTAAAGGTGTTTGATTAGGGTGTTAACCTGGTTCTTCTCAGTTTAATCCAGcagtgctggtggtgcccagtgggatctttagggaatgaatggaagccattcctgggtcaggtgcaagagacaccccggtccagacctaaggaaagccaaattgtagtcccaagtgtcaaatactgaactcagtcgtgtttagagctcatttcacccaaagtcctttgccagtgctaaagagtagttgccgagtagcatgtgctagcgtgtcagttaataactgcgctttccacagattgagaagctcgctcagactctgtcttgcgattacaatcaaccttcaccggaggttgtgagcgtggctgcctcatctcctacctcaagtcctcccactaagaagtgctccaaggctcaagctcaggcaagatggcagtggtctgccaagaaggctcttctgcagtgggctcaggagcagtgtgccaggtgagtagtttgctgtgccagatgagacagctgtgtccttccatccacacaacctcatggacttctggctctgacagtcgggctgatagtacactcccagcaacaccgtttcttagtgaatcttccagaagtggatcttggtgggcttggctgattctgctttgagaggtggcagctttaccctggttgctggctttccattgtttattgctgtgagCAGATAAAGAATATTTAAGTTTTCTAACTCCCCTTCCCTTTTTGAATGGGGGTCTCACTGTGTGGCTCTGGCTCTCCACAAACTCACACCCACCTACCTCTGCTTTATATATATGCTGGGAATAAAGGCACACACCAGCATGCCTGGCATTAAATATCTTTTTAAGGTACAGTTCTCCCATGC
This mutation is a 640 bp deletion beginning at Chromosome 12 position 75,887,919 bp and ending after 75,888,558 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
47173 | GTG GGC TTG GCT GAT TCT G | Wild type Forward | A | |||
47174 | ACA CAG TGA GAC CCC CAT TC | Common | A | |||
47175 | GCA TTC CAG TCT GCC TGT TC | Mutant Forward | A | |||
47176 | Fluorophore-1 | CTG GTT GCT GGC TTT CCA | Quencher-1 | WT Probe | ||
47177 | Fluorophore-2 | AAG GTG TTT GAT TAG GGT GTT AAC C | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
47173 | 0.40 uM |
47174 | 0.40 uM |
47175 | 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 |