Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr1:165187875-165188017 143bp GCCTCTTTGCAGTGTTTTACA AGAGCATCCTTCTCCCTTCC
Mut= 131 bp
Wt= 143 bp
Fam=Mut
Hex=Wt
Wt Sequence (deletion in lower case):
AGCACCATTTTTTTTTAAAGATTTATTTATTATTATATGTAAGTACACTGTAGCTGTCTTCAGGTTACCAGAAGAGCCACCATGTGGTTGGTGGGAATTGAAATCAGGACCTTCGGAAGAGCCGTCAGTGCTCTTAACTGCTGAGCCATCTCTCCAGCCCCCTCGAAGCAACATTTTTATTGGCCCTTCAGaccatctctaacacaattgtgttttgtttgtttgttttcagtaagtgtgcatgacgtatgtaattgccgtttttgaaactgcaggttgtggaggcgtcgtcgctgagctggggcaccagaataaaaggcttcatcgcatgctttgctctgggaatcctgtgctcagtgctggtaaggtttcctcccacccgctgcctttgcccagctctcagctgtagccttccctttggcccagtgacttttctctgtgacactcattacatggagtggataagaaggggagggaggcagggagggcagctatgtggctctggtgggagggggtcactgtgctcaagtgcatgtgtcctctgcttccccttaggggactcttctgctgtgggtgcccaggaagggactcggcctctttgcagtgttttacaccctgggtaacatcatgtcaattgggaggtaacctttttcttttctctttgcttcaactgtgctgtttattaaacaataaaatatctaccgcgtttgagGGAGGAAGGGAGAAGGATGCTCTGTTGTTTTTAAGGATTCAGAAAGAAGGAAGGTAGATACTTGGCGTATATCACATTCATATCTTATTTCAAAAACATCTTATGTCGTGTGTTTTCTCTGTGACACCCCTGGAAGAAAGCAGAAATGGTGTCACCAGTGCTTTCTATGAGGGAA
This mutation is a 521 bp deletion beginning at Chromosome 1 position 165,187,898 bp and ending after 165,188,418 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
46139 | GCC TCT TTG CAG TGT TTT ACA | Wild type Forward | A | |||
46140 | AGA GCA TCC TTC TCC CTT CC | Common | A | |||
46141 | GTG GTT GGT GGG AAT TGA A | Mutant Forward | A | |||
46142 | Fluorophore-1 | ACA TCA TGT CAA TTG GGA GGT AAC | Quencher-1 | WT Probe | ||
46143 | Fluorophore-2 | TTA ACT GCT GAG CCA TCT CTC C | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
46139 | 0.40 uM |
46140 | 0.40 uM |
46141 | 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 |