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Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr18:35650235-35650370 136bp CATGGTGGCCGAAATGAC TCAGGTGCTGGGATTAACG
Mut= 142 bp
Wt= 136 bp
Fam=Mut
Hex=Wt
Mut Sequence:
gctgctgcctccttactaaataaagcctgccttcctctcacatgcctgtcggccggagtccggagccccacctccacgctgcacctgtgttggagaaagttcggcatgggggcttcacccctaaaagcccacagcttaggtttctctgctgccaaggccgctagctcaggcgttgacgctggcgctttaagacgctttttaaaacaaaacaagacaaaatttaaaaaaaatcacttataccttgcatgatggtatacaccgttaatcccagcacctgagaggcagaagcaggtggatctctttgagttcaaggacagtcgaggctgtctcaaaaagtcaagtaaataacatttaaaat
This mutation is a 4975 bp deletion beginning at Chromosome 18 position 35,650,319 bp and ending after 35,655,293 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
52631 | CAT GGT GGC CGA AAT GAC | Wild type Forward | A | |||
52632 | TCA GGT GCT GGG ATT AAC G | Common | A | |||
52633 | TAG GTT TCT CTG CTG CCA AG | Mutant Forward | A | |||
52634 | Fluorophore-1 | AGT CCT AAT TTC CTG CTG CCA C | Quencher-1 | WT Probe | ||
52635 | Fluorophore-2 | CTG GCG CTT TAA GAC GCT TT | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
52631 | 0.40 uM |
52632 | 0.40 uM |
52633 | 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 |