Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr9:121780106+121780213 108bp CCTTAGGGGACAGGATGGTC TTTCAGGTCTTCACCCAATG
Mut= 106 bp
Wt= 108 bp
Fam=Mut
Hex=Wt
Wt Sequence (deletion in lower case):
ACAGGCCTCACAGCTGGAGGAGGAGAGGGGCAGCTCTCCCAAGCTGGCTGGGGGAAGTTTCAGAGGTTGCATAGTATCAAGTGTCCATCCACACAATGCAGCTGTTGGACAACTCTCCAAGGTGGGCTCTTTGTGCCTATAACCACTCGGTTAGgcagataaaaagggtggcatggaaggctatagattttaagggacttgggggggggctggcgggggggggaagccttgaacagttggtcaccaaagatccaaagccacagggggacctccctctcagagggggaaacttggctgatctgggccaggaggtgagcacaggaggtgagcacaggaggctcatggtggctgacccaaggatactgcctgcccacagtttgaccacttggactctgagtggctggggatgccgcctctcccttcacctcgctgcctcttcggcctgggggaggctctcaacgccatctacgtggtcggcggccgggaactcaaggacagcgaagacagcctggactcagtcctgtgctacgacaggctgtgagtgggcaggacggggccaccttaggggacaggatggtctggggcaggggccctagggtggtgctgggaccacagcagcaaccagGAGGCCTGCAAGGGTGCACAGCCATTGGGTGAAGACCTGAAAATAATCCTTGAGCAGAGGGGGAGATGGGCTGGCTCAAAGGCAGGAGAGCTCAGGGGAGGGGTTCCAGGAGTGTATTGAATGCAGTCTGCATGTGTCTTGTGGGTATGAGTGCAGAGA
This mutation is a 471 bp deletion beginning at Chromosome 9 position 121,779,701 bp and ending after 121,780,171 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
44116 | CCT TAG GGG ACA GGA TGG TC | Wild type Forward | A | |||
44117 | TTT CAG GTC TTC ACC CAA TG | Common | A | |||
44118 | ACA CAA TGC AGC TGT TGG AC | Mutant Forward | A | |||
44119 | Fluorophore-1 | CTG GGA CCA CAG CAG CA | Quencher-1 | WT Probe | ||
44120 | Fluorophore-2 | CCA CTC GGT TAG GAG GCC | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
44116 | 0.40 uM |
44117 | 0.40 uM |
44118 | 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 |