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Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr12:74302199+74302291 93bp TCAGGTAGCCGAAGACATCA CAGCTGTTGAGTTAGTTGGAAAG
Mut = 88 bp
Wt = 93 bp
Fam = Mut
Hex = Wt
MUT Sequence (junction in uppercase):
gattcgttccttcggaaatgctttggctggtcactgtgcctgacggttgtttgtcattagtgtccttaacCAtggttgctgtctttccaactaactcaacagctgtgcctgctgtttctt
This mutation is a 5723 bp deletion beginning at Chromosome 12 position 74,296,535 bp and ending after 74,302,257 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
51852 | CAG CTG TTG AGT TAG TTG GAA AG | Common | A | |||
51853 | TCA GGT AGC CGA AGA CAT CA | Wild type Forward | A | |||
51854 | ATG CTT TGG CTG GTC ACT G | Mutant Forward | A | |||
51855 | Fluorophore-1 | AGA CAG AGC GTT CTA TAG ATA TCA GCA | Quencher-1 | WT Probe | ||
51856 | Fluorophore-2 | TTG TTT GTC ATT AGT GTC CTT AAC CA | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
51852 | 0.40 uM |
51853 | 0.40 uM |
51854 | 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 |