Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr4:57647918+57648007 90bp TTGATGGAGAAGTCCTGCTG TTCCCGAAGCACTTTGGACT
Mutant= 95 bp
Wild Type = 90 bp
Wt Sequence: GACAGATTTGATGGAGAAGTCCTGCTGGAAAAGATGTGGACcaaaaggtgtcatctgttgtcttctctccttcccacagtccaaagtgcttcgggaaaaatggctgctgcagggtgtaccagcgggaacagcggaggaggaggaagccaggcgcaggcagtctgaagaagatgaattcaaagtcaagcagcttgaagataacattcagaggtaggtagttcccagcctgcagtaaccactgctctcctgaaggggacgctttcgccgtgctgcgacttttccctggggctgtagattgctgtcctaaatagatactggtgtcggctaacttttCAGTGTGAGTTGCTGTTATTCCTCAGCGAATAGTACAGAGATGGTTTCCCCCACCAATGAGGTCTTTATTATAAGCAGTCTGCCCTCCCCCAAATGCTCTCCTGTAAGT
Deleted Sequence: caaaaggtgtcatctgttgtcttctctccttcccacagtccaaagtgcttcgggaaaaatggctgctgcagggtgtaccagcgggaacagcggaggaggaggaagccaggcgcaggcagtctgaagaagatgaattcaaagtcaagcagcttgaagataacattcagaggtaggtagttcccagcctgcagtaaccactgctctcctgaaggggacgctttcgccgtgctgcgacttttccctggggctgtagattgctgtcctaaatagatactggtgtcggctaactttt
292 bp deletion beginning at Chromosome 4 negative strand position 57,648,243 bp and ending after 57,647,952 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
37627 | TTG ATG GAG AAG TCC TGC TG | Common | A | |||
37628 | TTC CCG AAG CAC TTT GGA CT | Wild type Reverse | A | |||
37629 | CTC ATT GGT GGG GGA AAC | Mutant Reverse | A | |||
37630 | Fluorophore-1 | AGT GTG AGT TGC TGT TAT TCC TCA G | Quencher-1 | MUT Probe | ||
37631 | Fluorophore-2 | AGG TGT CAT CTG TTG TCT TCT CTC | Quencher-2 | WT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
37627 | 0.40 uM |
37628 | 0.40 uM |
37629 | 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 |