Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr15:78956995+78957098 104bp TGAAACTGAATGCTGGAGGA TTCAAGAAAGTCACCATGAGC
Mut= 119 bp
Wt= 104 bp
Fam=Mut
Hex=Wt
Wt Sequence (deletion in lower case, and insertion ^G^):
GGCCAAGGCGCCAGGCCGGGCTGTAGATCTGACACCTCCTGCTGCCTGGCTCTGATCAGATTCTGGCTGTGGGGCAGGTGTGAAACTGAATGCTGGAGGAGGGTTGTTCTTTGGCCTGGATATAGCTGCCGACCCT^agcccttccttagcgcaggctggccttgctcatggtgactttcttgaatcctagagcactgctgcccacccagggcactgcctgggctacagcctctgccccagtcgccagactccagggtccccagggagactcccaccaggcctgctctcaggtgagccgaccggatcggatcgggttccttcggtttcatgtcccgcattgttcttggctgggccctggc^TTATGAAACAGTAATGAGGGGCCCTGCTCCCGAGGCTGGCCCCTCTAAGGTTTCTGCTGCCTGGAACACTGCAGCCACCGCGGCCCAGAGTCGGGGCCTGGGAGGGTGGCAGCGTGGGGGCCTGAGTTGGAGCCCCCCGGG
This mutation is a 223 bp deletion beginning at Chromosome 15 position 78,957,051 bp and ending after 78,957,273 bp (GRCm38/mm10). In addition, there is a single (G) bp insertion at the deletion site.
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
45089 | TGA AAC TGA ATG CTG GAG GA | Common | A | |||
45090 | TTC AAG AAA GTC ACC ATG AGC | Wild type Reverse | A | |||
45091 | CAG GCA GCA GAA ACC TTA GAG | Mutant Reverse | A | |||
45092 | Fluorophore-1 | CCT TCC TTA GCG CAG GCT | Quencher-1 | WT Probe | ||
45094 | Fluorophore-2 | CCG ACC CTG TTA TGA AAC AGT AAT | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
45089 | 0.40 uM |
45090 | 0.40 uM |
45091 | 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 |