Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr10:85995611-85995752 142bp GGGGAAACGTCTCTTAGAAGC AACATCCCTGGGCTTGTGAC
Mut= 148 bp
Wt= 142 bp
Fam=Mut
Hex=Wt
Wt Sequence (deletion in lower case):
CAAGACGTGCCTTGCGATTATTGTAGCATCCTTTAAAGGCAATTTTTCATCGTTTCCATCACAACATTGGACATGTAATGAAAACTTCTCACAGTAGCCCTGTGGTCTCAGGCAAGCTTCTGTGCctctcttaagactggagtttccgtatgtgaagccaagactacacctcttttgtggggagggggcatgctgcttcctcctctaacatctggtctttctcaatgctgtgtctgtagggttaaagatactgtgtcaccaccatcttccttatgatcaacaaacacttcttgcatccctcacaggtcttcaagttgggatgaaggtactggagcaattggcaaaggaaatcgtcatcccagatttgaacggttctgagtctcttaaatttctgaagattgactatgtaaagtacaacttctcaaagtaagagagtggagcatgttgacttatatcgttaggggctcaaaaccagtggagaaatctatggcagccctaacttagttatctttttttttttttctgtttcatctaagtcctgacttctattcacaagtttgcctcatagctcaatatatctaatactgtgtcgaccacctgatgcatatgtagctgaaaggggaaacgtctcttagaagctcagttcatttaagctaatttaacctcagccacattaattttctcatcTTTGAGGTCTTGTATGGGGCTCTAAGCTAAGCACATTTCATCTATACAACTTAGTCACAAGCCCAGGGATGTTTGGGGAAGGGGAAATGGACATTTTAATGGTAACAAGAATCCAATAAGATTACAAA
This mutation is a 562 bp deletion beginning at Chromosome 10 position 85,995,684 bp and ending after 85,996,245 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
45252 | GGG GAA ACG TCT CTT AGA AGC | Wild type Forward | A | |||
45253 | AAC ATC CCT GGG CTT GTG AC | Common | A | |||
45254 | CGT TTC CAT CAC AAC ATT GG | Mutant Forward | A | |||
45255 | Fluorophore-1 | AAG CTA ATT TAA CCT CAG CCA CAT T | Quencher-1 | WT Probe | ||
45256 | Fluorophore-2 | AAG CTT CTG TGC TTT GAG GTC TT | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
45252 | 0.40 uM |
45253 | 0.40 uM |
45254 | 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 |