Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr4:108266719-108266835 117bp TAGGGAAGCACTGGGACGTA CCCAAAAGAGAACTTTCCAAGA
Mutant= 114 bp
Wild Type = 117 bp
TTTGGTATCCAGATTGATAGAGACCAGACCTCATAATACCACACTTCTAGAGCTGAGAAGAGAGCATGGAGAATCACCATTGGCTCTCGGGTGCCTAATCCTACAGCGCCTTAGCGCTACCTGGCACGTGGTGCTGTCTGATAGTAGGGAAGCACTGGGACGTATCGCTGCCTAT^G^gtatctaatagtgaatctccgcaatgtctgttccattctgattctgctgttactagctccataatcttggaaagttctcttttgggtcttagtctagacaataatatcttcatctcaccaaattgtatcaagtaagtaaaatgcagctgcatggaattatgttagtgtggtgaaatttttgctcttctttgtctgtgattttgaagttaaaatgctgcttatttttccccaggtctcctgaatgatggaactgtgggaattttcagaggtaaccagatgcgcctaaagcgagcttgcattcgcaaagcaaagatttccgctgttgctttccggaaagccttttgccaccacaagctagtagaacttgatgccacgggtgtgaatgctgacatcactatcacagacatcataagtggccttggcagtaacaagtggatccagcaaaatcttcagtgcttagtactaaactcattaactctgtccctcgaggatccatatgagcggtgcttcagccggctctctggtcttcgagctctgagcatcactaatgtgctcttttacaatgaagatctggctgaagttgcttcgctgcctagactggagagcctggatatttctaacacctcaatcacagatatcactgccctgctggcctgcaaagacagactcaagtctctcaccatgcaccatctgaaatgtttaaaaatgactactacccagatcctggatgtcgttcgggagctaaaacatctgaaccatcttgacatctcagatgataaacagttcacgtcagacatagcccttcgattgttagagcaaaaggacatcttgcccaatcttgtgtccctggatgtttcagggagaaagcatgtgacagacaaagctgtcgaagcctttatacagcagcggcctagcatgcagttcgtagggttgctggctacagatgctggatactctgaattccttatgggcaaaggacacttgaaggttagatattaaaaattgtattttgcttgactggtcaggtttttttttcctagttccttaagacttcatTAGGGTAAATGTGTAATATTTGAAACACTCTGATACAAGGTATGGAAATAATTGTATAGTGGAGAAAGCCTTACATTAGGGTAATAATCCCAATTAAAAGTCCAGTGGGACCAAGCATGGCGAATGCTTTTAATCCAAGCACTCACAGAGCAGACGAAGGTAACTTCCTGCATGTACATAGCCTGGTGTACATGG
This is a 1055 bp deletion beginning at Chromosome 4 position 108,265,749 bp and ending after 108,266,803 bp (GRCm38/mm10). There is a single bp insertion (G) at the deletion site.
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
47463 | TAG GGA AGC ACT GGG ACG TA | Common | A | |||
47464 | CCC AAA AGA GAA CTT TCC AAG A | Wild type Reverse | A | |||
47465 | CCC TAA TGT AAG GCT TTC TCC A | Mutant Reverse | A | |||
47466 | Fluorophore-1 | AGT GAA TCT CCG CAA TGT CTG | Quencher-1 | WT Probe | ||
47467 | Fluorophore-2 | CTG CCT ATG GTA GGG TAA ATG TG | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
47463 | 0.40 uM |
47464 | 0.40 uM |
47465 | 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 |