Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr17:35520687+35520798 112bp TTCTTCTTCGCCTGATCTCAC TTCATCTACCACACCACTGTCA
Mut = 109 bp
Wt = 112 bp
Fam = Mut
Hex = Wt
Wt Sequence (deletions in lower case):
GAAGCCCTCCGATGTGCTTGTTTGACGTTTAACCCATCTGCCTGCTTATGTGTGGATCTCTGGAGAGATGGTGTTCTTCTTCGCCTGATCTCACTAGCCCTtaggtcgactgagcccagaaagtggatgaccacgtgtgcttagtagggacttggtggttgattgacagtggtgtggtagatgaatatatcctcctgcctcagctcagccccttgcttccaccctcccagcagctcaccttttcccctcctgtctttgaaggtctctggagctggggttctccagtgccctgagccagcaggccgagctgatctcacggcagctccaagagttgcggcggctggaggaggaagtccggtccctacgggagacctcgctgcagcagaagatgaggttggagactcaggccgtggagctggatgctctggcagtggcagagaaggccggccaagctgaggccgagggcctgcgcaccgccttggccggagctgaaatggttcggaagaacctggaagaggcgaagcacaaggagctggaggagattcagagcctgcaccaagagcaggtgaacacgggggtgggaggggcttagaagcatatcagggcacgccgtccagTAAATTGCAAGGAGCCTCGGGAGCAGCAGACCCGTGGAGGTGGGGGAAGCCCCCCAAAATACTTGGGTATTTTGGTTTCCCTCAGGCTGCACCTGTTACTTGGGAGTCAGTGAGCTCGTAACCTTCAATA
This mutation is a 516 bp deletion beginning at Chromosome 17 position 35,520,715 bp and ending after 35,521,230 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
50647 | TTC TTC TTC GCC TGA TCT CAC | Common | A | |||
50648 | TTC ATC TAC CAC ACC ACT GTC A | Wild type Reverse | A | |||
50649 | GGG AAA CCA AAA TAC CCA AG | Mutant Reverse | A | |||
50650 | Fluorophore-1 | CCA GAA AGT GGA TGA CCA CGT | Quencher-1 | WT Probe | ||
50651 | Fluorophore-2 | CCC TTA AAT TGC AAG GAG CCT | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
50647 | 0.40 uM |
50648 | 0.40 uM |
50649 | 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 |