Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr13:56176178-56176268 91bp TCCCTTGAGACCTTTGTCTGC TCCCGGTTCATTCATCTGTC
Mut= 99 bp
Wt= 91 bp
Fam=Mut
Hex=Wt
Wt Sequence (deletion in lower case;and insertions with carrots ^g^ (ggctg insertion):
CTTTATTCCTTGCTGAGAAGGAAGGGCACACCTGTGTTATAAAACAATACAATTTGGAGAGGATGGGCAGTTATGTCCTGTTGGAGTATAGGGAGATGGAATTCCAAGTTTAGTGTCAACCTCCACCCCATCCCAGCTTGGGGAGAATAACTGTATCTATTTACACATGGTCCTCATCAGCAAGATGTGGCCCCAAGATCTCTAAGTAGGCAGCTTGCCTGGGCTCAGAAAACTACAGAAGTGTGTCTTTGTCATGTTCTTCCCAGGATCTATGGAGAGGCCCCTCACAGTCCTGCAGGTGAGCCTGTACCACCCTA^cacagggcccggtcgcctttgcccacgtcccacagcagctgcaacatgacgccagccggctgttggttgggcgaggacagaacacccatctccagctgcagctgccacagttgtctcgctaccatttgtccctggaaccctacctggagaaaggcagcagtctgctggccttctgcctcaaggtgctgacccgcaagagctgtgtgtgggtcaacgggctgccactgaggtacctggagcaggttcccttgggcaccatcaacagaatctccttctctggcatccagatgctagtccgcaaagaggggggagcctcccttgagacctttgtctgctacttccatctcagcccttcacccctgatttac^AGACCCAAGGCTCAGGAGACAGATGAATGAACCGGGAAAACTCTGTTAAGAAAGAGACTCCCCCTGTTTTGGGGGAGGCAACTCATGAACTCTCGAGGTCTCTCCACAGCCCCTCCCAGTCTCAGACAGCTGCTTCCCTGCCAAGCCCGGAAGAAGCAATAGAAACAAAGTTGCAGAAGGGGCCCTCAGACAATGCACCCCACTAGCCCTGCTGCTTGAGAGCAAACCTGACAAAAATAGGGTTTGAGATGGTAGAGCTATATCCTGTGAGGTTGACACACAGAGTTCACATGTAAAGCCTGCTGGCCGCCATGTTCAC
This mutation is a 368 bp deletion beginning at Chromosome 13 position 56,176,215 bp and ending after 56,176,582 bp (GRCm38/mm10). In addition, there is a 5 bp insertion (GGCTG) at the deletion site.
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
52824 | TCC CTT GAG ACC TTT GTC TGC | Wild type Forward | A | |||
52825 | TCC CGG TTC ATT CAT CTG TC | Common | A | |||
52826 | TCC CAG GAT CTA TGG AGA GG | Mutant Forward | A | |||
52827 | Fluorophore-1 | TCA GCC CTT CAC CCC TGA | Quencher-1 | WT Probe | ||
52828 | Fluorophore-2 | ACC ACC CTA GGC TGA GAC CC | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
52824 | 0.40 uM |
52825 | 0.40 uM |
52826 | 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 |