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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.
>chr2:73318403+73318497 95bp CACTTTTGGGCATGGACCT GAAAATGGATGTGGGTTGGT
Mutant= 90 bp
Wild Type = 95 bp
Wt Sequence: CACTTTTGGGCATGGACCTTGTCAGgtaacagttgtgctgctttcccactgtagatgatgtgggaaatGGtaaataccaacccacatccattttc
Mutant Sequence: agtgctcttaaccactgaaccatctctccagcccctgcatgagttttattattgaaaatttaaTGtaaataccaacccacatccattttc
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
36697 | CAC TTT TGG GCA TGG ACC T | Wild type Forward | A | |||
36698 | GAA AAT GGA TGT GGG TTG GT | Common | A | |||
36699 | AGT GCT CTT AAC CAC TGA ACC AT | Mutant Forward | A | |||
36700 | Fluorophore-1 | CAG GTA ACA GTT GTG CTG CTT T | Quencher-1 | WT Probe | ||
36701 | Fluorophore-2 | CCA GCC CCT GCA TGA GT | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
36697 | 0.40 uM |
36698 | 0.40 uM |
36699 | 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 |