Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr19:23378945-23379073 129bp GACCCAGTGTTCAGGCAATAC GAGGATCAAAGACAAGGAATTAGTC
Mut= 125 bp
Wt= 129 bp
Fam=Mut
Hex=Wt
Wt Sequence (deletion in lower case; and insertion with carrots ^g^ (T insertion):
CTCAGCCTGTTGTCAAATGGGGAAGTGGTATGGTCAAACCGTGTAGAGGTGTCCAGCCTTTGACACTGTCGTATGATATCCTTTACAAATGTGTTGGGATGCATTAATATACTATAGGATGCATGAGGCCCACAGGGTATGGTTAGATGTGGCCCTTTCACAGGAGACCAGGGACCCAGTGTTCAGGCAATACCG^ttgggatgatgcaatgcaagctctgtcttcctacagatttatgggaccaaacagttgtcctttctccaggttgcatcagaagactaattccttgtctttgatcctctccctttcaggccattatgtctatatggacacctcttttgccaggcaaggggagaaagctgtgctgctgagttctgatttgcaggctgaggagtggaactgtttgcgcctggtctaccagataaccacacctccgggatctgtgtcagaccccagccagctgaacctctatgtgagatttgaggatgagagcttcgatcgcttgctttggtccactaaggagccttcagacagctggctcatagccagcctggatcttcaaaacagctccaagaaattcaaggtaggtggggtttaagaggaagatgtgagggtgtgtaacatgtgttctatgctatatg^AGGTCTTAGAGAAGTGGTTCCAACCTTCTGAATGCTACAACCCTTAAATACAGTTCCTCATGTTGTGGTGATTCTCCAACCATAAAACTTTTTTTTGCTGCTACTTTATAACTGTAACTTTGCTACTGTTATGAATTGTAACATAAGTTTCTGTGTTTTCTGAGGGTCTTAGACAACCCCAATGAAAGGGTCATCTA
This mutation is a 446 bp deletion beginning at Chromosome 19 position 23,378,605 bp and ending after 23,379,050 bp (GRCm38/mm10). In addition, there is a single bp (T) insertion at the deletion site.
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
47645 | GAC CCA GTG TTC AGG CAA TAC | Common | A | |||
47646 | GAG GAT CAA AGA CAA GGA ATT AGT C | Wild type Reverse | A | |||
47647 | GCA GCA AAA AAA AGT TTT ATG G | Mutant Reverse | A | |||
47648 | Fluorophore-1 | ATG GGA CCA AAC AGT TGT CCT | Quencher-1 | WT Probe | ||
47649 | Fluorophore-2 | CGT AGG TCT TAG AGA AGT GGT TCC A | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
47645 | 0.40 uM |
47646 | 0.40 uM |
47647 | 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 |