Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
305 bp deletion beginning at Chromosome 5 position 117,979,701 bp and ending after 117,980,005 bp (GRCm38/mm10).
Mutant= 83 bp
Wild Type = 86 bp
>chr5:117979673+117979758 86bp CTATGTGTCTGCCAGGGTCA GAAGCCATTACAGGGGACCT
Sequence (deletion in uppercase) tgtaagagccgctgtcactgctctatgtgtggcctccctcccttgggactctcagctatgtgtctgccagggtcagccacgctAACTCATGGTTGAGGCCTCCTGTGTGTGTCTTCTCCACAGGTCCCCTGTAATGGCTTCAGTGACATTGAAAACCTTGAGGGCCCAGAGATCTTTTTTGAGGATGAGCTGGTGTGTATCCTAAACATGGAAGGAAGGTAAGGCACAGCTTCACCAGGATAACTGCAGAGGGAATTGCTGTGGGGCAGAGAGAGAGAGAGAGTGAGAGAGAGAGAGATTGCGCGAGCGAGCGAGGGAGCTGCGGTGTTTCCAGATAGTTTATGTTATGCCACAGTTATGCTCTGAGAAGCTTTGTGTACACCTACTCccggggtgcacagttgacaggtggctcacccctgaagtcccagtgctcaggaggtcggggcaggagaagcggcagctttgagagagcgctgcatagtgagcttcagaaca
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
40603 | CTA TGT GTC TGC CAG GGT CA | Common | A | |||
40604 | GAA GCC ATT ACA GGG GAC CT | Wild type Reverse | A | |||
40605 | ACC TCC TGA GCA CTG GGA CT | Mutant Reverse | A | |||
40606 | Fluorophore-1 | CAT GGT TGA GGC CTC CTG TGT G | Quencher-1 | WT Probe | ||
40607 | Fluorophore-2 | CAC AGT TGA CAG GTG GCT CAC CC | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
40603 | 0.40 uM |
40604 | 0.40 uM |
40605 | 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 |