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.
>chrX:168670520-168670657 138bp GCTAGCAAAGCTTCACAGATCC CACACAAGCTAATGATCTGAAAGTG
Mutant= 155 bp
Wild Type = 138 bp
Wt Sequence: gctagcaaagcttcacagatccttcctcttggatcattttcttcttgagacctccagggcctTGccaccactgatgaacccaacagtgaaccataggatcctagctggtgaaacactttcagatcattagcttgtgtg
Mutant Sequence: gctagcaaagcttcacagatccttcctcttggatcattttcttcttgagacctccagggcctTCcacaagctatgtatttttttaagtacgattgtgaactatacttcataatgactaatttttaatggtacataacagagattggcttctgcc
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
34423 | GCT AGC AAA GCT TCA CAG ATC C | Common | A | |||
34424 | CAC ACA AGC TAA TGA TCT GAA AGT G | Wild type Reverse | A | |||
34425 | AGG CAG AAG CCA ATC TCT GT | Mutant Reverse | A | |||
34426 | Fluorophore-1 | AAC CCA ACA GTG AAC CAT AGG A | Quencher-1 | WT Probe | ||
34427 | Fluorophore-2 | CAG GGC CTT CCA CAA GC | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
34423 | 0.40 uM |
34424 | 0.40 uM |
34425 | 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 |