Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr11:78263023+78263123 101bp GAGCTACTTGGAGGACTCTGC CGGCCTCTTGAGAAACAAG
Mut= 102 bp
Wt= 101 bp
Fam=Mut
Hex=Wt
Wt Sequence (deletion in lower case):
CCGTAGTTCTGGTTGTTGTGCCTAAATGAGTATGACTAGAGAAATATTTTAGGAAAATCGTAAAGTTCCTAAGAAAAATCCGGCTTCAGGATACCACCCCTTGCAAGGCCTTTCTAGAATCAAAAAGGgtagagggtcttataatccaactgttttttttctgcaggtggcttgtggtccggttggccaccaggtggtgtcagcgcaagttgcaggcagagctaaagattggctccttccgatttttttggatccagaatgtcagtcttaagtttcagcagcaccagcagacggtggtgagttccgggaattgcagagagaaagagagagagagagagagaggagaagggcgggagtgtgtggtgtgtgtgttggtttggagggggtttcaaactttagcttcctccatgggtagttttcctagttaactccacccttgttgttgttgtttttgttgttgttattattattattgtcaagaggatcttaggagctacttggaggactctgcaatagatgccagttgtttcaagtgtttatggGACTAGATCTGGTGGTATAAACCTGTAATTCTTGTTTCTCAAGAGGCCGAAGCAAGAGAATTATAAATTCAAGCTCTGCCTAGGCAACTTAGTGAGTGCTTCTCTCAAAATTAAAATGAGCTGGAATAGTGGTTCAGTGGTA
This mutation is a 414 bp deletion beginning at Chromosome 11 position 78,262,661 bp and ending after 78,263,074 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
43348 | GAG CTA CTT GGA GGA CTC TGC | Wild type Forward | A | |||
43349 | CGG CCT CTT GAG AAA CAA G | Common | A | |||
43350 | AAA TCC GGC TTC AGG ATA CC | Mutant Forward | A | |||
43351 | Fluorophore-1 | AAT AGA TGC CAG TTG TTT CAA GTG T | Quencher-1 | WT Probe | ||
43352 | Fluorophore-2 | AGA ATC AAA AAG GGA CTA GAT CTG G | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
43348 | 0.40 uM |
43349 | 0.40 uM |
43350 | 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 |