Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr2:104042846+104043011 166bp ACTTGCGTCTCGGGTGTGT CCACTAACTTTTGCCCGTTG
Mutant= 180 bp
Wild Type = 166 bp
The WT Forward primer (primer 60656) anneals over the nucleotide sequence containing mouse genomic variations rs219604498
The Mut Forward primer (primer 60658) anneals over the nucleotide sequence containing mouse genomic variation rs224815574
The Mut Reverse primer (primer 60659) anneals over the nucleotide sequence containing mouse genomic variation rs260031365
Wt Sequence (deletions in lower case):
CCCAGCAAGTGAGGGGCAGTTGCTTGGAAGAGCATCACTTCTTTTGGTATTTGTAGGTTTTTCTCTTGTGACAGCTAGAATGCAGGCCATGTTTTCTAATGTGCAATGGAGAATTCAACTTAGTGGCTTCTCACAGTATGAATCTACATCTATCTACTGCCTTCCACTTCCCATAAAGGGTTCAAGTTGAACCTCTAGAATGACGTCAGCTGAGAGAACTTCCAGAGCTGTTTTTAAATGAAGTATTGAATATATCAGGTTTGGAAAAGTGAGACAAGAGCTTTGTTATAATGTAATAAATTAATTGTTCATTTCCTTGGTGCCCCTTTCCAATTCTTTGTGCCAGTGTTTTTTTCCcacgttgagaggaaacaaacctcttttgtttagtgtttaaaattattttccaattcagtgtctggtattttgacagtatattttaaacggaattcacttgtcataagatggtttcatgggttgacagctgacctagactaccctgaagtctgagtgtgcgcacacacttgcgtctcgggtgtgtatttgcgcacacacttgtgtctcgggtgtgtatttgcagagggcgcacgtgaggaagacctggatgctgtcgaagctcagatcggttgcaagctcccggatgattatcgctgttcataccggatccacaacgggcaaaagttagtggtgccggggtaaggtgtttgatgttttagtaattttcagaaatgggtattactttctagaatgaatcaaattacattttattcatagtttttcttagctatctttactaaacatgtataagaaatgtatgtatgggggggttccttccttttccttatagCAGTCCATAATGTAATATGTATAGATTAAGTTAAAGTTTACCAAAATAAATGTGACTTATAAGAGCATTATTCCTGCCAGCACATTTTTATGATTAAAGTAAGATCTGGTTGAGAAGGAAGCTGTGGGTGCAGGAGGGGATGTGCATGTGTGCATATTTGTGCATGTGTGCATATTTGTGAGTGCATTTGACTGAATGGCTTGATGTGGTG
This is a 490 bp deletion beginning at Chromosome 2 position 103,873,026 bp and ending after 103,873,515 bp (GRCm39/mm39)
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
60656 | ACT TGC GTC TCG GGT GTG T | Wild type Forward | A | |||
60657 | CCA CTA ACT TTT GCC CGT TG | Wild type Reverse | A | |||
60658 | GTT TGG AAA AGT GAG ACA AGA GC | Mutant Forward | A | |||
60659 | GCT GGC AGG AAT AAT GCT C | Mutant Reverse | A | |||
60660 | Fluorophore-1 | ATG ATT ATC GCT GTT CAT ACC GGA | Quencher-1 | WT Probe | ||
60661 | Fluorophore-2 | CTT TCC AAT TCT TTG TGC CAG TG | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
60656 | 0.40 uM |
60657 | 0.40 uM |
60658 | 0.40 uM |
60659 | 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 |