Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr5:130035050+130035150 101bp CCTGATGGAGCTTGGAGTGA GGGATTTTCGGCAGAGTACC
Mut= 110 bp
Wt= 101 bp
Fam=Mut
Hex=Wt
Wt Sequence (deletion in lower case):
AGGGGTTCAGTGCTGTCTTCAGGCTTCTGTGCACACTTAGGCATAAACACACCAGACATACGCATCCGCACACCTGTGTAGTACATTCATATACTCTCATAGGCACAAGCATACACATAAGTAATATTAAAAAAAAAAGGAAAGTTGTCTCTATCTGAAATTGctaaccagggcagaacttaaggcatttctagtttaggttgttcctgagcgagtgagaaagctgggattaactagagggttcatttgtggacacttaaccttgaaatggatgtgaactgatgacggctctctgttcttttgtaggaaggatgcgaacgcagcactgctcagcaactatgaggtgaggacccccacgcgggccattgtgttttcctctctggtttagctttgcttttgaagacgttggattggttttctttagaaacgagagggcaggtggcccctgatggagcttggagtgatatgagtttggtgaactacacatggttcttcagggaagCGGGGAGGCTCTGGTTCACAATAGGTACTCTGCCGAAAATCCCAGAGTCTCTAGAGCAGGCAGATGTCCTAGCAGAAGGCTGCTGCTTTAGAGCGCAGTCCTGCTGTAAAAGTCTCCTATTAAATGCGTAGCTTCTTGATAATAACCATTCTCTACTTGTCTTTGATTCT
This mutation is a 339 bp deletion beginning at Chromosome 5 position 130,034,769 bp and ending after 130,035,107 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
43037 | CCT GAT GGA GCT TGG AGT GA | Wild type Forward | A | |||
43038 | GGG ATT TTC GGC AGA GTA CC | Common | A | |||
43039 | TCA TAG GCA CAA GCA TAC ACA | Mutant Forward | A | |||
43040 | Fluorophore-1 | CTA CAC ATG GTT CTT CAG GGA AG | Quencher-1 | WT Probe | ||
43041 | Fluorophore-2 | CTC TAT CTG AAA TTG CGG GG | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
43037 | 0.40 uM |
43038 | 0.40 uM |
43039 | 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 |