Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr12:81485273-81485363 91bp AGTTACAACTGATGAGCCACGA CAGATAAAGGGCGGGGTTG
Mut= 91 bp
Wt= 91 bp
Fam=Mut
Hex=Wt
Wt Sequence (deletion in lower case):
AGTAGGATAGAGGGTTTGCGGAGCAGAAACCGGGACGGGGGATAGCATTTGAATTGTAAATAAATAAAATAATAAAAAATTTTAAAAAGTTCAGTTACAACTGATGAGCCACGACTTAAAATGTGTTCATTAACCTGGTGctacttgtgcattagcttaaaaaccaaccccgccctttatctgagtttcttgagtcgcagaagtcaagatccagaatatgcgaattacagattttccaaatcgaaaaacccaattgcacccggcgtatcctgaggtcacggaaccagagctcagaagcctcgacttccgggggagggaggaagcagaagtgcgttaaggccaatcgtttccgtttgcctcggggaggcttatcaggcgtggacctggcacccaaagcctgcgctgagtctacaacttcgcctgagacgtgttgcgggccgcgctgggctcgctctggaagcggctgactaagcaggccgcaccttctgccatgattgcgcccgctgtgttgcgagctctgcgtaagaacaagactctacgctatggagttcccatgttggtgagcggctgtgacgggagcaggggctgcgagtccggaaggggcgggccctctttggctgagtggcgtattggtgctgtctttcggatactgctgactagggggtctgtgggagccaccggagctttgaggtttttgtttttgtttttaagttcacgctcaaaagtgtacctcgggtcccgccatcccccaccccccgtttcctttcccttcgtgaacgcgcgtgTTACTATCCCCGTTTTACAGTACCTTTCCTGGGTTAAATGTTGAGATTTTGCAAGAAGACTAATCAAATGTCTTCTTTAATAATTACATTCATTTGTAGTAATTATGTTCATAATTTGTTGTTTG
This mutation is a 645 bp deletion beginning at Chromosome 12 position 81,484,671 bp and ending after 81,485,315 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
45359 | AGT TAC AAC TGA TGA GCC ACG A | Common | A | |||
45360 | CAG ATA AAG GGC GGG GTT G | Wild type Reverse | A | |||
45361 | CAA CAT TTA ACC CAG GAA AGG | Mutant Reverse | A | |||
45362 | Fluorophore-1 | CTA CTT GTG CAT TAG CTT AAA AAC | Quencher-1 | WT Probe | ||
45363 | Fluorophore-2 | AAC CTG GTG TTA CTA TCC CC | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
45359 | 0.40 uM |
45360 | 0.40 uM |
45361 | 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 |