Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr15:25895129+25895234 106bp AAGCCTCACTGAAAGGTAGGG ATTCAGCGATATGCCTCTGC
Mut= 109 bp
Wt= 106 bp
Fam=Mut
Hex=Wt
Wt Sequence (deletion in lower case; and insertion with carrots ^g^ (GTATA insertion):
TGAAGACTGCACCATGCCAACCTCCCCTCCCATACCATTGCCATTACCTCGACTTGAGCAGTCATGTTTCTGTCCTTTCTGAAGTGTGATGGTTTCCACCTCTTGTCAAATTTCATACTGACTCCTTGACAGCACTTTTTTCTTAAGTGGACATGTTTTCTTCCCACTGGGCAGGACTGTGTTTCGGAGTGTCCATCCTGCCTTTGTGATACGTAC^tcaagggttttatagactatcacctgcatccttcgcaatgatgtacgcagatactgtatgggttgacagtgtctagcacctgagatgtgagcattccgcttgcacaagtgtgggaggttgctaagctggcaaatgctcctggggagtgacagcttgctcacctttcacattttgctttccgttgcaggtgcccagctgtggagaagcctcactgaaaggtagggagtttatccttcaacaatgtggcttatggatggcagggcagtgg^CGTGGGGAGGTGATTTGAGTTGCAGAGGCATATCGCTGAATTCATGTTTTAGCGACAGTGACCTAAGTCCCTATAATGACTTGCAAGAGTGTGCAAAGTGAAGCTGAAGCTGAGGACAAGACGGGGAAGTCACTCGAGGGATAAATGCATTTCTCAGAGTGGCTGGGCAGCGGATTTGTGCGGGAGGATGCAGCAGGGAGGGAGAGGGGCCTTTCTGTTTGATGTCTTCTCTCAGATTC
This mutation is a 268 bp deletion beginning at Chromosome 15 position 25,894,926 bp and ending after 25,895,193 bp (GRCm38/mm10). In addition, there is a 5 bp (GTATA) insertion at the deletion site.
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
47522 | AAG CCT CAC TGA AAG GTA GGG | Wild type Forward | A | |||
47523 | ATT CAG CGA TAT GCC TCT GC | Common | A | |||
47524 | GGA CAT GTT TTC TTC CCA CTG | Mutant Forward | A | |||
47525 | Fluorophore-1 | CTT CAA CAA TGT GGC TTA TGG A | Quencher-1 | WT Probe | ||
47527 | Fluorophore-2 | CTT TGT GAT ACG TAC GTA TAC GTG G | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
47522 | 0.40 uM |
47523 | 0.40 uM |
47524 | 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 |