Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr2:167048317-167048420 104bp TCTGGAGTAGGAGGGGTGAG GCAGAATGAAATGCCAGGAG
Mut= 97 bp
Wt= 104 bp
Fam=Mut
Hex=Wt
Wt Sequence (deletion in lower case):
AAGCCACCAGTTCAGTGCCTCCCTACCTTGCTAGTGCTGTACAGCATTAAGTGTTAAAGCCAGAGTCCTCTGTCCGTTTGTCCAGGTCCACAGCCTTGCTCTTAAAAACATTACACTTCTGTGCTCGGCAGCCCAAATTTTATCAGTAGCCTGCTCTTACAAGTGCAGTGAATGTCCTGTAAGCACAACTTTGCTCCCTGAGgcaccagttgagataggattacacacacaactctcacctcagatattgcacggttttataggcatctacgggtgtcagaagaccagcattgtgcgagtgggcggaaggagcaacagtgaaatcctgaagcagttcaccctgagggagctgaggaacaagcgggagttccgccgcaccctgcccatgcacctgcggagggcctatatgagtgtgagtcagagttctggagtaggaggggtgaggggcccacacagcatagcagccacttTCTAGCAGGCTCCACAGCCCTCCGCAGGACCCAGCCTCCTCCTGGCATTTCATTCTGCTCACCTTGTGGTGCCCAAGACAAAGGGTGAAGAAAGCTACCAGGCCCTTCAGTCTGTCCCTCCACACTACCACAGTGTGGAGCCTGCTCGCTGGGAAGTGGCTTGCTTTTTCCAGACAGGTTGTCCTCATATTCACTCTGTA
This mutation is a 268 bp deletion beginning at Chromosome 2 position 167,048,375 bp and ending after 167,048,642 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
47552 | TCT GGA GTA GGA GGG GTG AG | Wild type Forward | A | |||
47553 | GCA GAA TGA AAT GCC AGG AG | Common | A | |||
47554 | TGC AGT GAA TGT CCT GTA AGC | Mutant Forward | A | |||
47555 | Fluorophore-1 | CAC ACA GCA TAG CAG CCA CT | Quencher-1 | WT Probe | ||
47556 | Fluorophore-2 | CTC CCT GAG TCT AGC AGG CTC | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
47552 | 0.40 uM |
47553 | 0.40 uM |
47554 | 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 |