Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr17:83421489+83421642 154bp TCTGCTTCCCTAACTAAATCTACTCAC CAGGACATAGAAATAGCTTCTCG
Mut= 145 bp
Wt= 154 bp
Fam=Mut
Hex=Wt
Wt Sequence (deletion in lower case):
GTAGCTGGCGGGTGTCAGCTAACTCTGTGCCCTAGCTACCCCGGTGCTGGTCGGACCGAAAGGGGCTTATGCCCCTACTCAGGCCGGGTTTTCTGCTTCCCTAACTAAATCTACTCACTATTTCTAAACACTACAAACTGAATTGTAAAACTCTGCTCCttaaggaatattttgaaatttgataacttttcttttttaactattttaggccaaccaagccttcgagaagctatttctatgtcctgtataaccaatggaagtggtataagcagaaaacagaatcataccagctctgtatcaatagcaagaaaagaaactctttcttcagctgctaaaaggtacctgatagtaatggggtggggacactggccctgaACAACTGTGCATGATGATCTAGGTACTCATGAGAAGACTTCCAAGTAGAGCTCTGTGGGAGTCAAGCACGCATGCCTTGAATTCCTTTTTTTGTTGTTGTTTTTTTGGTTTTTTGTTTTTGCGAGACAGGGTTTCTCTGTGCAGTCCTGGCTGTCCTGGAACTCACTCTGTAGACCAGGCTGGCCTCAA
This mutation is a 216 bp deletion beginning at Chromosome 17 position 83,421,557 bp and ending after 83,421,772 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
39883 | TCT GCT TCC CTA ACT AAA TCT ACT CAC | Common | A | |||
39884 | CAG GAC ATA GAA ATA GCT TCT CG | Wild type Reverse | A | |||
39885 | AGG CAT GCG TGC TTG ACT C | Mutant Reverse | A | |||
39886 | Fluorophore-1 | TTT AGG CCA ACC AAG CCT T | Quencher-1 | WT Probe | ||
39887 | Fluorophore-2 | TGC TCC ACA ACT GTG CAT G | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
39883 | 0.40 uM |
39884 | 0.40 uM |
39885 | 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 |