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Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr11:120795185-120795265 81bp CACCAGTGTCACCTTGATCCT CCACTGGCACAGACTTGTAAA
Mutant= 89 bp
Wild Type = 81 bp
Wt Sequence: caccagtgtcaccttgatccttctccgtctcagtaccctccTgtcgagaaccaacagttctttacaagtctgtgccagtgg
Mut Seq: caccagtgtcaccttgatccttctccgtctcagtaccctccTCatggggagagttaatccctgcctctgactgtg
A 434 bp deletion beginning at Chromosome 11 position 120,794,790 bp and ending after 120,795,223 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
65223 | CAC CAG TGT CAC CTT GAT CCT | Common | A | |||
65224 | CCA CTG GCA CAG ACT TGT AAA | Wild type Reverse | A | |||
65225 | CAC AGT CAG AGG CAG GGA TT | Mutant Reverse | A | |||
65226 | Fluorophore-1 | TCT CAG TAC CCT CCT CAT GGG | Quencher-1 | MUT Probe | ||
65227 | Fluorophore-2 | CTC CTG TCG AGA ACC AAC AGT T | Quencher-2 | WT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
65223 | 0.40 uM |
65224 | 0.40 uM |
65225 | 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 |