Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination. The transgene genotype is determined by comparing ΔCt values of each unknown sample against known homozygous and hemizygous controls, using appropriate endogenous references.
Mutant= 130 bp
Wild Type = 149 bp
>chr7:120429483+120429631 149bp ACAGAAACATTGACTCTCATGCT CACACCCAGCTTAAGGCACA
Wt Sequence: tgagagaggcatccctctgtgtctgtataaggtcactagaaagaagttgcttttgttctgacttttgatcactattgagcaatacagaaacattgactctcatgcttgaagagtgatactgaaaatgggattttagtactgaagagatgggttataATtaatta
attgttagcaagttggtcagaaaatttcaagggatgtagattaagacatgtgccttaagctgggtgtggtggcacacgcctttaatcccagcacacaggagg
Mutant Sequence: GGTGAGAGAGGCATCCCTCTGTGTCTGTATAAGGTCACTAGAAAGAAGTTGCTTTTGTTCTGACTTTTGATCACTATTGAGCAATACAGAAACATTGACTCTCATGCTTGAAGAGTGATA
CTGAAAATGGGATTTTAGTACTGAAGAGATGGGTTATAAgaattccgaagttcctattctctagaaagtataggaacttcatcagtcaggtacataatataacttcgtataatgtatgctatacgaagttattaggtggatccttaattaatt
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
33524 | ACA GAA ACA TTG ACT CTC ATG CT | Common | A | |||
33525 | ATG TAC CTG ACT GAT GAA GTT CC | Mutant Reverse | A | |||
33526 | CAC ACC CAG CTT AAG GCA CA | Wild type Reverse | A | |||
33527 | Fluorophore-1 | AGA ATT CCG AAG TTC CTA TTC TCT AGA | Quencher-1 | MUT Probe | ||
33528 | Fluorophore-2 | TGG TCA GAA AAT TTC AAG GGA T | Quencher-2 | WT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
33524 | 0.40 uM |
33525 | 0.40 uM |
33526 | 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 |