Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr19:5638040+5638170 131bp CCTTGACTCAGTGTCCCTCCT AGTCAGATACTTTCTTCCTTACACG
Mut= 140 bp
Wt= 131 bp
Fam=Mut
Hex=Wt
The Common primer (primer 53932) anneals over the nucleotide sequence containing mouse genomic variations rs238833930.
Wt Sequence:
ggcggggcgggctgcgagggggtctgactcagttacccctctcagtggagggcgccttgactcagtgtccctcctgggtgggggtacgggtgaatcttgactccgtttcctccggctccccccacccccgtgtcgatgcgattagttctggcttcctttccgtgtaaggaagaaagtatctgactcagtttccccagaagaggagaggagggtattaccc
Mutant Sequence:
CCCAGCGGGGCTGCTAAAGCGCATGCTCCAGACTGCCTTGGGAAAAGCGCCTCCCCTACCCGGTAGAATTCGAAGTTCCTATTCTCTAGAAAGTATAGGAACTTCCTCGAGCGTGTCGATGCGATTAGTTCTGGCTTCCTTTCCGTGTAAGGAAGAAAGTATCTGACTCAGTTTCCCCAGAAGAGGAGAGGAGGGTATTACCCCCTCCTCAGTGTCACTTATGAACGTAAAACAGGCACTGACTCAGTTTCCCCTGGCAAAGGAATGCCTCTGTGTCCCCTTTCTCTAGTTCCCCGGCCTCTGTCAGGGCAGGTTTCAGGGTCTTGGCTCAGTTTTCTTGGAAGGGAGGGAGCTTGGCTCTGTCAGGCCTTCGCAGGTGAGGGTGGGCACTGGAGTTTATTGACAATGTGCCTTGTCTTTCCCTTTC
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
46629 | CAG ACT GCC TTG GGA AAA GC | Mutant Forward | A | |||
53931 | CCT TGA CTC AGT GTC CCT CCT | Wild type Forward | A | |||
53932 | AGT CAG ATA CTT TCT TCC TTA CAC G | Common | A | |||
53933 | Fluorophore-1 | ACG GGT GAA TCT TGA CTC CG | Quencher-1 | WT Probe | ||
53934 | Fluorophore-2 | CCT CCC CTA CCC GGT AGA ATT C | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
46629 | 0.40 uM |
53931 | 0.40 uM |
53932 | 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 |