Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr15:57995327+57995463 137bp CGGATCATACAGGTGAGTACTGGT GTGGCCAGAAAGAAGAGCTG
Mut= 131 bp
Wt= 137 bp
Fam=Mut
Hex=Wt
Wt Sequence (deletion in lower case):
TCCTTTCTGGGGCCCCACCCTCCTGAGCTGATTACCTCCAAAGCCCAGCCTCCCAGAACCACTGCACCAGAGACTGTGTCTCAACATAGAATTGGGGGTGCATGTTCAGCCGTAGCGCGTGTTTATCAGTGAATACCCAGttagtaagtgacgccctcactttgcgtccccagaacatctccatcaggagtgtggaaggggaggtgtattgtgccaagtcaggcaggaaatttgccggccaaatccaagagaagttcatcatctcagactggaggtttgtcttatccggatcatacaggtgagtactggtttcatctgctgtccaggcttccgggcagtgatgggctaagtggggtaccagcTGGATGATGGCTGCTCCCACCCCACCCCCCCCCAGAGCTCCCAGCTCTTCTTTCTGGCCACAGCTAGAGCTCACCAAATAAACGAAGCCAGAAGTGAGAAATGTGGCCCCTTCCTGACACTGAAATTGGACAGAGACTTCTTTCTACTCTCTCCGGC
This mutation is a 222 bp deletion beginning at Chromosome 15 position 57,995,181 bp and ending after 57,995,402 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
42291 | CGG ATC ATA CAG GTG AGT ACT GGT | Wild type Forward | A | |||
42292 | GTG GCC AGA AAG AAG AGC TG | Common | A | |||
42293 | AGA CTG TGT CTC AAC ATA GAA TTG G | Mutant Forward | A | |||
42294 | Fluorophore-1 | CGG GCA GTG ATG GGC | Quencher-1 | WT Probe | ||
42295 | Fluorophore-2 | AAT ACC CAG TGG ATG ATG GCT | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
42291 | 0.40 uM |
42292 | 0.40 uM |
42293 | 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 |