Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
Mutant= 117 bp
Wild Type = 137 bp
>chr11:100713810+100713946 137bp GGAAGATAAGACAGCGACTTGA GCATCCGAGAGTGCAACTG
Wt Sequence (deleted region lower case): GATGGAGCTGTAACCCCGCACATTCAGGTGATCCAGTTCCTTCATAGCCTCCTTCCTGCCCTCTGATGTGAGAGAAGGGCCCAGGGAGAAGCAGAAGCCGGTTCTGGACAAAAGCTGCAGCGCTTGCAAGGAAGATAAGACAGCGACTTGAGTGTTTTTGCCCTTCTGGATGGGAAGAGTGGgcgggcccttcctcttcgcacccgccccggatggggggttggggagccctcgcgccctcaggcagcagttgcactctcggatgcagcgggtaggaagcagtttctccaccgggcagcgcgaacccggggaaaaccgggtagcttcccgatgtcccagcgtggcccttgctgaacggaatcaagtggccactctaccagtgaggctgctcagggatgaggtgcagggcaatggctgtgagcaaccaagtttccaaataaaggtggatgcccaaggtttcgctccggaggacttggtgGTGCGGATAGACGGCCAGAATCTGACGGTGACCGGCCAGCGGCAACACGAGTCGAATGACCCGAGCAGGGGCCGCTACCGCATGGAGCAGAGTGTTCACCGACAAATGCAGCTCCCGCCGACCTTAGATCCTGCAGCCATGACCTGCAGCCTGACCCCCTCTGGCCATCTGTGGCTCAGGGGACAAAACAAGTGCCTACC
296 bp deletion beginning at Chromosome 11 positive strand position 100,713,863 bp and ending after 100,714,158 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
37116 | GGA AGA TAA GAC AGC GAC TTG A | Common | A | |||
37117 | TCG GGT CAT TCG ACT CGT | Mutant Reverse | A | |||
37118 | GCA TCC GAG AGT GCA ACT G | Wild type Reverse | A | |||
37119 | Fluorophore-1 | TCT GAC GGT GAC CGG C | Quencher-1 | MUT Probe | ||
37120 | Fluorophore-2 | CCC TTC CTC TTC GCA CC | Quencher-2 | WT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
37116 | 0.40 uM |
37117 | 0.40 uM |
37118 | 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 |