Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr9:99640090+99640200 111bp CCTCTGACTTGTGCCCTTCA GCCCCATTTATACAGTCAGC
Mut = 123 bp
Wt = 111 bp
Fam = Mut
Hex = Wt
Wt Sequence (deletions in lower case):
ATCAGGATTTCTGTTTTCTTTCCCTTCCCACTCCCACCGTTCTTTCTGTCTTTCTGTGGTGTTGGGAATTGAACCTAGGGTCTCACCCACTCCCGACCCTTCAAATTCTTTTTTATTTCTGAGGCAGCCCATGGACCCTGTGGAGGCAGAGTTTCACTACAGGATATTTccacgctgtctgggggatctaatccagcccttccaattgcttttcttctgtgtagtgccacttgtgtgacaagaccttcatgaatgccacctttctccggggccacatccaacgcagacatgcaggcatggcagatgtcggtgagtccaggcttcattggggaactgctgggcaaggatttggtgtgtgaggcccatgcacatagacctgctcttagagcctcttagcgattccacatggtgccctgtgcagtcttgcctgtgtaggggacaagaggcatcagacaggtaggtttagttttagttttccatgaacagtggccagtacttagcctctgacttgtgcccttcactgtgtgtggagcaggcagtgactcataagggcctttagcctccctgtaGCCTCATCTGTAGCAATCCGAGGCTGACTGTATAAATGGGGCAAATGAGCAGTCAGACCAGTTCTCTGGCTGTTCTGTGTACGGAGCTTATCACGACTTAATCACTTTATTTAATTGTACCATTTCAGATTCCAAAAGGAGTAACAA
This mutation is a 390 bp deletion beginning at Chromosome 9 position 99,639,764 bp and ending after 99,640,153 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
49330 | GCC CCA TTT ATA CAG TCA GC | Common | A | |||
49331 | CCT CTG ACT TGT GCC CTT CA | Wild type Forward | A | |||
49332 | ACT CCC GAC CCT TCA AAT TC | Mutant Forward | A | |||
49333 | Fluorophore-1 | AGC AGG CAG TGA CTC ATA AGG G | Quencher-1 | WT Probe | ||
50768 | Fluorophore-2 | AGC CCA TGG ACC CTG TGG | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
49330 | 0.40 uM |
49331 | 0.40 uM |
49332 | 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 |