Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr9:71548658-71548739 82bp ATTCTGACAAGAACAGGTGAGG CACACACACAACCAGTGTGC
Mut= 82 bp
Wt= 82 bp
Fam=Mut
Hex=Wt
Wt Sequence (deletion in lower case; and insertions with carrots ^g^ (A insertion):
GGTTTGCCTAGAGTGTTTTTTTCCAGATCCATACATATGTCTATGCATTGGGCCCCTTTGAGTTCTGAGCTCTGACCTAGTGGATGTGTGAACAGGGAGTGTTGTACAGACTTGGAGAGCTGGGTGTGCCTGGTG^ttcatcctctgcctagctccatctggatccattgattatgtctgttttaggaacggcatcagctccagctccagctccttgagcatgagacagaaatgtcaggggagatggcagattctgacaagaacaggtgaggcaggctggccttggacttcgagcaactcag^AGGTGGATATGCACACTGGTTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGCATGCGCGTGTGCACTAGATGCACATGTGTGCATGTTCTTGCCCACAGAGGCCAGAGGAAGAAGTCTGGTGTCCTTCTCTTATCACTTTCTACCTCATTCTCTTGA
This mutation is a 166 bp deletion beginning at Chromosome 9 position 71,548,688 bp and ending after 71,548,853 bp (GRCm38/mm10). In addition, there is a single bp A insertion at the deletion site.
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
42924 | ATT CTG ACA AGA ACA GGT GAG G | Wild type Forward | A | |||
42925 | CAC ACA CAC AAC CAG TGT GC | Common | A | |||
42926 | TGT GTG AAC AGG GAG TGT TG | Mutant Forward | A | |||
42927 | Fluorophore-1 | CTG GCC TTG GAC TTC GAG | Quencher-1 | WT Probe | ||
42928 | Fluorophore-2 | TGG AGA GCT GGG TGT GC | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
42924 | 0.40 uM |
42925 | 0.40 uM |
42926 | 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 |