Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr14:21686322-21686410 89bp CTCATATTCCCTCCCCAGGA ACCAGGTTTGAGAATCACTTTC
Mut= 95 bp
Wt= 89 bp
Fam=Mut
Hex=Wt
Wt Sequence (deletion in lower case):
CAAACCCATTAATGTTTGGAAGCTACAGAACAGGGGCCTCTTTTAAAGGTTCTCCCAGGGGCTCTGTGAAGCAGGCTCAGAGGAACAGATTGTGTTTCAGTTGCCTTTGGGGAGATCTGACAGGTCCACCTCCAGGACCTGCAGCGTAAGGTGAATGATCcaaagggaaagggcaaatgcccagactgcgcctacccagggctgggcagatggtagctttcttatagtcatgaaaccaaacggtgcccagtggcctggggctcgggaagagcgctgctttgggtctttgcctgtaatctccctgcgattactgggtccagcgttgaaagccgtgcaccacccaaagcgcggcgtccaccaaccgaccgacccttctccctgcgcccagggccacggcgctggaccgctatgggctgcagaaggcgggtttcacgcacgtgctgaacgcggcgcacggccgctggaatgtggacaccgggccggactactaccgagacatggccatcgagtaccacggcgtcgaggccgacgacgtgcccaccttcgacctcagcatctttttctactccgctgcggccttcatcgactcggcactccgagatgaccacagtaagtgaccctctccaactctgccctgacaaggccccgcccccaggctgaaaacccacccatcaggtcccgctcccagacataaaacacgccccccctccccgccagccgaggcaccacccctcccgcccacagtgtgctttccccctgctaggtcatgccccaaggcatgctatcctctcccctctggggagaggtcctgccctcatattccctccccaggatcttccaacccggccctgccccagtggatcAGAAGCAGGATCTACAAGAAAGTGATTCTCAAACCTGGTGTACCGGGGCTGAGGATTGAAGGCAGGGATTGGCTGAGCTAACTGCCTGGCCATGTGTGTTGCTGGTGGGGTTCGGGCTTGATATACAGTGTTGAGATAGGAAAGGGGGAG
This mutation is a 703 bp deletion beginning at Chromosome 14 position 21,686,361 bp and ending after 21,687,063 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
45420 | CTC ATA TTC CCT CCC CAG GA | Wild type Forward | A | |||
45421 | ACC AGG TTT GAG AAT CAC TTT C | Common | A | |||
45422 | CTT TGG GGA GAT CTG ACA GG | Mutant Forward | A | |||
45423 | Fluorophore-1 | CTT CCA ACC CGG CCC | Quencher-1 | WT Probe | ||
45424 | Fluorophore-2 | CCA GGA CCT GCA GCG TAA | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
45420 | 0.40 uM |
45421 | 0.40 uM |
45422 | 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 |