Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr2:25966388-25966469 82bp GTCTGACAGGCATTCCTCTGTTC GCAAACCTCACCATCTGACC
Mut = 105 bp
Wt = 82 bp
Fam = Mut
Hex = Wt
Wt Sequence (deletions in lower case):
CCTAAACAAAAGCTGTTTGGTCTCTGCTTGGTACCTGACCTTGACTTTGGAAAGAAGATAAGCAGAGCTCCCTTCTCAGAATGAATGTCATTGTACCATAGCCCCttctttaatccgatggcatcttggtttcctttgagctctctagcatcccaggaaatttgtgcttgcgtcccaccatcattcagattatgctggcctttgcacaaatgcctcattgtgcctctgtcgtgctaatgaggctgttagttgaccctagatctttgtgtgttgtgtgtggtaccttggctgggattttgctggacggtgcttttgtgtgttctcatctggctggtgatgcttgagactggactgtgcatgtccattcacccagtgctctctctcctgccatagacaacatccctgaggacctccgagacccgttttacatcgaccagtatgagcaggagcacattaagccacccgttatcaagcttctcctgtccagtgagctgtattgccgtgtctgcagcctcatcctaaaaggggaccaggtggctaccttgcaaggacaccagtctgtcatccaggccctgtcccggaagggcatctatgtgatggagagtgatgatacccctgtgacagatgctgacctcagccaggcacctattaagatggtgagtgttgccgggcgtggtggcgcatgcctttaatcccagcacttgagaggcagaggcaggtggatttctgagttcgaggccagcctggtctacaaagtgagttccaggacagccagggctatagagaaactctgtctcgaaaaaccaaaaaaaaaaaaaaaaaaaaaaaaccaaaaaaaaaaaaccccaaaaaacaaaacaagatggtgagttgtctgacaggcattcctctgttcccttggctgagcaggcaagactcagtgcccttccatTAGGGTCAGATGGTGAGGTTTGCCAATGTGTATTGGTGGACCTTAATGACAGACCTGCATTTTAAAGAATGTCATCATTCTGGAAAGAACAGCTTGCTTTTGGG
This mutation is a 826 bp deletion beginning at Chromosome 2 position 25,966,411 bp and ending after 25,967,236 bp (GRCm38/mm10). There is a single bp (T) insertion at the deletion site.
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
49900 | GCA AAC CTC ACC ATC TGA CC | Common | A | |||
49901 | GTC TGA CAG GCA TTC CTC TGT TC | Wild type Forward | A | |||
49902 | TGC TTG GTA CCT GAC CTT GA | Mutant Forward | A | |||
49903 | Fluorophore-1 | CTT GGC TGA GCA GGC AAG A | Quencher-1 | WT Probe | ||
51297 | Fluorophore-2 | AGA GCT CCC TTC TCA GAA TGA ATG T | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
49900 | 0.40 uM |
49901 | 0.40 uM |
49902 | 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 |