Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr17:25201236+25201339 104bp GCTTCATCCGAGAGTTCTTACA CACTTGGCCACCTGTTTCCT
Mut = 80 bp
Wt = 104 bp
Fam = Mut
Hex = Wt
Wt Sequence (deletions in lower case):
GGTAGTGTTAGTGAGGTGACAGACCAAGTGGCTCTGGAGCCATGGATTCTGCTGGCTTGCCCCACCTCCGGGAACCctcgggctataactgctgtactgtgctgttggttgcagggagcttcaggctcaggaagccctgcagaatggccagctcagtggaggggatggtgtacctgatctgcagcctggggtcttggccagccaggccatgatcgagaagattcttggtgaggaccctcggtggcaaggtaagcctggggttgctggagtccttgcaggggcgagccatggcttttctccaggtacctgcttcatccgagagttcttacactagagaagaggacagatgggaaagtcaggtttctcagcccactgCTAACCGTGAGCCATTTAGGAAACAGGTGGCCAAGTGGGATTGGAACCTAGCCGCCT
This mutation is a 299 bp deletion beginning at Chromosome 17 position 25,201,004 bp and ending after 25,201,302 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
52397 | CAC TTG GCC ACC TGT TTC CT | Common | A | |||
52398 | GCT TCA TCC GAG AGT TCT TAC A | Wild type Forward | A | |||
52399 | CTG GAG CCA TGG ATT CTG CT | Mutant Forward | A | |||
52400 | Fluorophore-1 | AGA AGA GGA CAG ATG GGA AAG TCA | Quencher-1 | WT Probe | ||
52401 | Fluorophore-2 | CAC CTC CGG GAA CCC TAA C | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
52397 | 0.40 uM |
52398 | 0.40 uM |
52399 | 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 |