Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr4:45301038+45301195 158bp CCACCAGTGAAAATAAAATAGC GAGCACCAGAAAGGAAAATGA
Mut= 148 bp
Wt= 158 bp
Fam=Mut
Hex=Wt
Wt Sequence (deletion in lower case):
TGGTGGGGTGAGGGTGGTGTTCTCTCTGATTACAACCTGGAGAGGGATATTATTCAGAGAGAACTTTTGCCTTGCGTACTCAAGGCCCTGGGTCAGATCCCAAGCGATGCACACAGTTATAATAATTCTGTAATGTTCACGGTGAATACCCACCAGTGAAAATAAAATAGCAAACCATCAtagtggtaaaattaaaaataaacacaagccagttgcaggcttgttttaaagacaaaaaccttttcttctttaactttatatataaatgaaaaaaggtggcgggcattcattttcctttctggtgctcatgaagttctcaaacaattttgttttctggataatggaagaaaaacatgcaaagaaaacagagacactgggaaaggatagtctcctcaaagaagagcaaaaggaagcaggaaagagaacgaaggaaagccaagcgtgcagaagaccccggtaacggtgagcgtggttgccggtgaaaggtcatggctttcagcaaacttgtttttatgttgctgggagagtagggcctattactcagttcctctgcaagtcagcggaccagtgTCAACTTCGAGGTTGCTTTTAAAGTAGAAGCTAAGTGACCATGTGCAGCTGTAATCTCAGAAACGGGATGACGCCGGCGGTAGCGCATGCTCACTCATGCTCTGTGCTAGCTTAAAATCATGCATTGAGTACCTCAGGGCTGCTACGTACTGAGATTATCCGCTTTGTGTAGA
This mutation is a 390 bp deletion beginning at Chromosome 4 position 45,301,069 bp and ending after 45,301,458 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
47481 | CCA CCA GTG AAA ATA AAA TAG C | Common | A | |||
47482 | GAG CAC CAG AAA GGA AAA TGA | Wild type Reverse | A | |||
47483 | TTT TAA GCT AGC ACA GAG CAT GA | Mutant Reverse | A | |||
47484 | Fluorophore-1 | ACA CAA GCC AGT TGC AGG C | Quencher-1 | WT Probe | ||
47485 | Fluorophore-2 | CTC AGA AAC GGG ATG ACG C | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
47481 | 0.40 uM |
47482 | 0.40 uM |
47483 | 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 |