Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
Wt Sequence (deletion in lower case):
TCTCTTGGTCCCTTTTATTTCTATAAATGCTCTGTGTTTTAAATTTCAAAATTAAGTTTTCATGTGGTGCTACTATTTTTCTACTTACTATTCCTGGCATGACGGTTGGATGCGTTCTTCCTGACATTAAGCAGTGATCATATTGTGAACTgcactggcactattattcaccctgctgtagtctgctgcccggtgacctgcctgtgccctcttttgcagataaacgcggtacgtgcgatagttcccaataagagcaacaatgagatcatcctggtcttgcagcactttgacaactgtgtggacaaaacagtgcaagcgttcatggaaggtaacgccagttccctcagtgtagactctggaacctgagccgcagcgcagtctccggacggcgctgcatactgtggtcttctgttagctagggtactcctttcattcccaagctatagaaaactggaagagatgctaagaaaactcctttgctgtcagatatgttctccccctccccaaatcaagaaactgtgcagctgacagactgtgggatttgaatGGAATTGGATAAAGTAATTGAAGACGCTATTGCAAAGGAGGAAGTCAAGTGGCCCACGAACACAGACCAGTTCAAGACCCAGCATTGATTTCCAAGCTCCATTATGAGTTAGTTTGGTTCTGGCAGGGGACTCAAATGTTATGAGTTCAGTTTCCTTACATTTCTATATGCTAGATAGAAAAAAGTAACAGCCTTGTCCAACTCCTTAGATTTATAGAGTTAATAAGTGCTTCTTAAAATTCTTTCCATATAACTTGTTTGAAAAAACTTTACTCTGAAGCTGTTTTGCCTTTGAACAGAGTGGCCAGGGCTCCTTGATGCGTTTTCCTGAATCCGCAAGCATAGAAT
This mutation resulted in a 396 bp deletion beginning at Chromosome 15 position 99,173,334 bp and ending after 99,173,729 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
44431 | TAT TCC TGG CAT GAC GGT TG | Common | A | |||
44432 | AGC AGA CTA CAG CAG GGT GA | Wild type Reverse | A | |||
44433 | CCT TTG CAA TAG CGT CTT CA | Mutant Reverse | A | |||
44434 | Fluorophore-1 | TGA ACT GCA CTG GCA CTA TTA TTC | Quencher-1 | WT Probe | ||
44435 | Fluorophore-2 | TGA ACT GGA ATT GGA TAA AGT AAT TG | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
44431 | 0.40 uM |
44432 | 0.40 uM |
44433 | 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 |