Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr11:5959180+5959305 126bp CTGTTTCTAAGGTTAAGAGGGAATG TCCTGCACACTAGGAAATATAACACA
Mut = 119 bp
Wt = 126 bp
Fam = Mut
Hex = Wt
Wt Sequence (deletions in lower case):
TACTGTTTCTAAGGTTAAGAGGGAATGAATAAAACCAAGAGAACCTCGGAGGTCACTTTCCActgtgttggggacagtgtgaccgaaagaaggttgtaaatatgtgttatatttcctagtgtgcaggaattcatgacctttacaagtcaactgattgtggaacgctcggggaaaggcagcagagcttctgtcaaagaacaaggtaagaggactcccgcccctctgagtgctggcctggctgtcacacatgctgcccgtgccgtgtgactgctcccaggcctgtgctcctctgctaccgcatgtcctcgccTTGCTTCTCCTATCCATAAAGAGCACTAAACCAAATGTTGAAGTCCACACTTGCTGCACCCTGATGTGCAACTGCACTTAGACAGCTTACACTCACTGCTCGTTAGACTCTGGCTCTCCACCTGCCCCAGCCTGGCA
This mutation is a 248 bp deletion beginning at Chromosome 11 position 5,959,240 bp and ending after 5,959,487 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
51301 | CTG TTT CTA AGG TTA AGA GGG AAT G | Common | A | |||
51302 | TCC TGC ACA CTA GGA AAT ATA ACA CA | Wild type Reverse | A | |||
51303 | GTG CAG CAA GTG TGG ACT TC | Mutant Reverse | A | |||
51304 | Fluorophore-1 | ACA GTG TGA CCG AAA GAA GGT TG | Quencher-1 | WT Probe | ||
51305 | Fluorophore-2 | CCA TTG CTT CTC CTA TCC ATA AAG A | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
51301 | 0.40 uM |
51302 | 0.40 uM |
51303 | 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 |