Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr5:115238266+115238412 147bp TGGGCTTTGCAGGTACGAG GCACCTTCTGTTCAGTTTTCG
Mut= 147 bp
Wt= 147 bp
Fam=Mut
Hex=Wt
Wt Sequence (deletion in lower case):
CTAGAAAAACGCATTAAAAACCTTAACCCTCTGTTCTTCATTTTAAAACATAATCCTCTATTCTTAACAGAAAACAGAGGGCTGGGTGTGGTGGGACGCGTTGGTAATGCCAGCGCTTCGTAGGAAAAGGCAGGGGATCAGAAGTTCGGGGTCGTCTTGGCTACACAGTCAGTCTGGGGTTAGCTTGAGGAAAAAAAGAAACCAGGAAGAGAGGGAGGGAGGATACTATATTGTTTTTGGAAACCACTTGAAAAGAACTCCCCTCCacaaaactgtcgaaatcccctcaaagaattcgcgtatttggctgtatgggaaaacctgtgctctgacttttaggtcccagatcgtgcgacactctcggactttaagtgcagggctgaccttaaacgttcacttgggggactctgagcgcacagaacttgtgtggaaaatccccagcgccggccggaggcgaacgcatgcgctcttacgcccgccgcgggaggcggagcgtggagattgacagggcacgctgcctgtccgagcctagtgccgccttcctcccgcatccggcgcggacgccatggtgcggttcaagcacaggtaagcagcgctgtccccgcgccgtccccgcgacccgggccgcgctcaccccgctgtcgccgcaggtaccttctgtgcgagctggtgtccgaggacgcgcgctgccgtttgagcctggacgaccgcgtgctgggcgggctggtccgcgacacgatcgcccgggtgcacggggccttcggcgccgccgcctgctccgtgggctttgcaggtacgagggcgctgcggatgcagtgcggggcgcgctttgccagggcgaggcccggccagtgtgtcgcgttttgtacgatagtggAGGGTTCTAGAAAATCGGAGTGCGGCGGGTCGAAAACTGAACAGAAGGTGCCGATCGCGCAGTGGGACAAGGCAAAAAGTTCAATACCCAGCCCCCGGTGTGTGTGCCGGGGTGGGTTGAGTTGGGGGATTCCGTTTACTGCCCGTTTTAGTGGAAGATGGGTGTTGAATTCACTAGGCTAAATGAAAGTAGCCATGTAGAATTCCTAAGAGCCATGATTTGACAGGTCACGTTAGCACGTTTTAAAATGTCAAATGTATCCCTCAAAGTTTAAACCCGGCTGCGAGCTCGAACA
This mutation is a 611 bp deletion beginning at Chromosome 5 position 115,237,751 bp and ending after 115,238,361 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
52786 | TGG GCT TTG CAG GTA CGA G | Wild type Forward | A | |||
52787 | GCA CCT TCT GTT CAG TTT TCG | Common | A | |||
52788 | AGT CTG GGG TTA GCT TGA GGA | Mutant Forward | A | |||
52789 | Fluorophore-1 | CAG TGT GTC GCG TTT TGT ACG | Quencher-1 | WT Probe | ||
52790 | Fluorophore-2 | AAG AAC TCC CCT CCA GGG TTC | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
52786 | 0.40 uM |
52787 | 0.40 uM |
52788 | 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 |