Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
Mutant= 112 bp
Wild Type = 116 bp
>chr9:80435486-80435601 116bp TTAACACAGGCTAGCACACTTG AGTTAGCCATGCAAAATGGA
Wt Sequence (deleted region in lower case)
CCAGTGTAGACAGACAATCCCTTCTTGATGTTCTCTGGTTCTGCTTTGTGTCCATTGGCAGTTAACACAGGCTAGCACACTTGTAAATTAGTGTGTGAACATAagctggaaaacactttctgtattgtgacactgtgtgctttagggaacagggttttccattttgcatggctaactttaactgttttgtgttacagtatgtcaagaagtcgtgtgggaagcatatcgtatctttctggaccgaattcctgacacagaggaatatcaagactgggtcagcctctgccagaaagaaaccttctgcctctttgacattgggaaaaacttcagcaactcccaggagcacctagatcttcttcagcaggtaaaccttttcaatatcctaggagcagcagggccccagcccctgccccttTGAAATGGTTACTAAGGCATAAGAGTTACTTGACAAAATACTTAGAGATAGCTGATAAGACAGACAGGAATTTTATTCCTGCAAAATAATATTGCACAGGCAGGTTGACGTGTAGTTGAAGGCAGAAAGAATGAAAATAGACTTTAGCTC
312 bp deletion beginning at Chromosome 9 negative strand position 80,435,559 bp and ending after 80,435,248 bp (GRCm38/mm10)
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
37133 | TTA ACA CAG GCT AGC ACA CTT G | Common | A | |||
37134 | TTC CTG TCT GTC TTA TCA GCT ATC TC | Mutant Reverse | A | |||
37135 | AGT TAG CCA TGC AAA ATG GA | Wild type Reverse | A | |||
37136 | Fluorophore-1 | TGG TTA CTA AGG CAT AAG AGT TAC TTG A | Quencher-1 | MUT Probe | ||
37137 | Fluorophore-2 | ACA CTG TGT GCT TTA GGG AAC A | Quencher-2 | WT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
37133 | 0.40 uM |
37134 | 0.40 uM |
37135 | 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 |