Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr12:86988091-86988183 93bp TCCTGCCCACTTCAATCAG AAACTTGGGGTTGGGCTAGG
Mut= 97 bp
Wt= 93 bp
Fam=Mut
Hex=Wt
Wt Sequence (deletion in lower casen):
CCTCCCTGTACCTCTCCAGACTCCAAGACAACAAGGACTTAGCCAAATGCAGTAGGTCTAGCCAGATGCTGACCAACTGTGGTCTCTGGGTCTCTAACCTGCTTGGTTCCAAACTAGGGAAACTGGAAAGGGTGATCCAAGGGGCAGGGTTTGGGAGATTTGTGTCCAGCCCTTCgaaccttggacaatgctgtaaggcttcctctctccttgtgccccttaggaatgtgatccaggatgctggccttgcggctgctcaacgtggtagcccccgcctactttctttgcatttccctggtgaccttcgtactgcagctcttcctcttcctgcccagcatgcgtgaggaccccacagccaccccgctcttctcgcctgctgtgcttcacggggcgctcttcctgttcctctcagccaatgccctgggcaattacgtcctggtcatccagaactccccagacgacctgggcacctgccaggggaccatgtcccagagacctcagtgcccaccgcccagcacccacttctgccgagtgtgttcccgagtcacgctgaggcacgaccatcactgtttcttcaccggcaactgcatcggcagcagaaacatgcgcaacttcatcctgttctgcctctacacctctctggcctgcctttactccatggtggctggagtggcctacatctcagctgtcctttccatctccttcgcccaccccctggccttccttacgctcctgcccacttcaatcagccagttcttctccggtgagtgggccatggcaggCAGGTTCAGCAAACAAGACCTCCTAGCCCAACCCCAAGTTTTTCAAATAAAAACCACAGGATGCTGGACTCAAACAGTCAGATAGACAGTTGTTTGGTATGACTATGTTCCAGTATTCCAGGAGGCATACTAAGAGAACAGATTATAATCTATCAGCAATTAGGATGAACTGGGCA
Thsi mutation is a 607 bp deletion beginning at Chromosome 12 position 86,988,132 bp and ending after 86,988,738 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
52511 | TCC TGC CCA CTT CAA TCA G | Wild type Forward | A | |||
52512 | AAA CTT GGG GTT GGG CTA GG | Common | A | |||
52513 | AAA CTG GAA AGG GTG ATC CA | Mutant Forward | A | |||
52514 | Fluorophore-1 | CCA GTT CTT CTC CGG TGA GTG | Quencher-1 | WT Probe | ||
52515 | Fluorophore-2 | CCA GCC CTT CCA GGT TCA G | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
52511 | 0.40 uM |
52512 | 0.40 uM |
52513 | 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 |