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.
Wt Sequence (deleted region is in lower case and insertion is indicated with ^^)
ATCATATATTTTATGTGTGTGTGTGCATACATGTATACCGCAGAATATATTTGGAGGTCAGAGGGATAAACACTTTAAAAATTTTAAAGTATTTTCAACAACATGGGAGTTAGTTTTCTCCTTTAATCATGCAGAGTCCAGGAACCAAACTCAGGCCACTAGACTTCAATAGCAAGTGCATTTATCTGTAGAGCCACCCTAATTTTGGAGGATTATTTTATTTTGGATAATCTAGAAAATAGTTTCTATATTAGAGCATTTTTTATTCTTATTTCATTAGCATATCCCATTGTGCTCA^ATTGTAAAAGGGTCAGTTGAAGTTATTA^tcaataggccttcttattaattcctatttataatttattaattcctatttataacttcaactgacccttttacaatttttttttgaaaaatatttaatatcctttaatcttttttttaatcttcaggtcagcccaagccagaggtgacttggtataagaatggtcaagccatagacttgggaggcactgtttccagttatgagttctttgagaaccagtatattcatttgttgcatctctcgtggtaagtggcttggcctctgtgaactactcactaaaattctgtcttccatggtgtgctcatgtttgcctcagaagtcctggactttgatgttactgtcaggcaacagcctaaccagccagtactgactaaagtgttccctctgtatgaatggattacctgaatatttaatgaaccgtgaggctgaaaatatttttaagaattgtattgggattagggatggttatcttcactccctagacagtatagcacACTAGTAATTTACCTATAATTTATTTCATATTAACATGGGGGTGATTTGTACAAACAGTATATCATATTATATAGGAAAGAGACTTAAGTATCCTCAGGGCTGATTATCTGGGAACTGAGATCTCCATAGGACAATGAAGAGTGATCATATTCATTAAGCTAAGTAACTATTTGGCCTTCTCTAGTTTACCATTGATAGACTTAAACTTTATCTAGAATTCTAGGGTT
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
34075 | CAT TAG CAT ATC CCA TTG TGC | Common | A | |||
34076 | TTG GGC TGA CCT GAA GAT T | Wild type Reverse | A | |||
34077 | Fluorophore-1 | CAT CAA TAG GCC TTC TTA TTA ATT CC | Quencher-1 | WT Probe | ||
34078 | Fluorophore-2 | TTT CAT ATT AAC ATG GGG GTG A | Quencher-2 | MUT Probe | ||
34079 | GAT AAT CAG CCC TGA GGA TAC TT | Mutant Reverse | A |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
34075 | 0.40 uM |
34076 | 0.40 uM |
34079 | 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 |