Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr4:139607713+139607822 110bp ACTGATGAGATGAAGCGCATGT CCAAACTCTTTCAGAACAGGTG
Mut= 117 bp
Wt= 110 bp
Fam=Mut
Hex=Wt
Wt Sequence (deletion in lower case):
TGGGGCTGCTTTTTCTGGTGATGGGTGAGCTGAGCTGGCCCAGTCACCAGGCAGGACTTGGTCCCTGTGTCTGTGTTCTCCACCTGCATGCCTCACACCTCACGCTTCCTGCCAATGCTGCCTGCTTCGTAGAAGACCTATGCAACTCCCTGCCGTGGTGGCTGGTCCCAGCCATATCCGTCCACTCCTCgggacagtggttccctcccagacttaaccccctctgcaccccagccaagcacccccgttaccttggtctttccttccccgtccaggtggtccctaagaagaaagatcgtaaggtggcttcggatgaagacatctcggagcaggacggggaggtgaatcggttttcagatgaggaggtcggctccatgaacatcactgatgagatgaagcgcatgtttaaccagctgtgagtatccaggttgcccagccagGGGACATATCCTCAGCTAGGCTGAGACAAGTCACCTGTTCTGAAAGAGTTTGGAGTGTGTGTGTGTGTGTGTGTGTGTGTGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGCACACTCGAGATCACACAAAAACCAGTTATCAGTCTGCGTGTCATTTCTCAGGC
This mutation is a 250 bp deletion beginning at Chromosome 4 position 139,607,520 bp and ending after 139,607,769 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
44131 | ACT GAT GAG ATG AAG CGC ATG T | Wild type Forward | A | |||
44132 | CCA AAC TCT TTC AGA ACA GGT G | Common | A | |||
44133 | TCG TAG AAG ACC TAT GCA ACT CC | Mutant Forward | A | |||
44134 | Fluorophore-1 | AGT ATC CAG GTT GCC CAG C | Quencher-1 | WT Probe | ||
44135 | Fluorophore-2 | CGT CCA CTC CTC GGG AC | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
44131 | 0.40 uM |
44132 | 0.40 uM |
44133 | 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 |