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.
Muant= 182 bp
Wild Type = 166 bp
Wt Sequence:
aaggggtggggactgccCTTTCTCACCCACCCTTGGGCGGTGGCAGGGCAGAGCTGGGGAGCACTGTCTCCTCCTCTTTCCCCTGCAGCTGCTCATTTCAGCTGGGTGGACC
CGAGGCTGCAACTCCGTGGGCTCCGGGAGGAGTGGATCTGCTCCGGCCAGGATGCCTGCGGCCACCGCCGGGATCctCTTGCTCCTGCTCTTGGGGACGCTCGAAGG
CTCCCAGACTCAGCGGCGACAGTCCCAAGCGCATCAACAGAGAGgtacagtccagggcgccctttccctggagcgctgctcctaacaggcttcagctagagagaaagaattgctggttggggaccactttctcg
ctttactttagggtgtgctcttccaggcgcaagggactgaactcctgcaaattaagagtaagacccccccgagagagctgt
Mutant Sequence: CCTCTTTCCCCTGCAGCTGCTCATTTCAGCTGGGTGGACCCGAGGCTGCAACTCCGTGGGCTCCGGGAGGAGTGGATCTGCTCCGGCCAGGATGCCTGCGGCCACC
GCCGGGATCCacagcaaggagctgctcaagcgcgatcccgtcgttttacaacgtcgtgactgggaaaaccctggcgttacccaacttaatcgccttgcagcacatccccctttcgccagctggcgtaatagcgaagaggcccgca
ccgatcgcccttcccaacagttgcgcagcctgaatggcgaatggcgctttgcct
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
11358 | GTT GAT GCG CTT GGG AC | Wild type Reverse | A | |||
28836 | CTC ATT TCA GCT GGG TGG AC | Common | A | |||
28837 | AGG CGA TTA AGT TGG GTA ACG | Mutant Reverse | A | |||
28838 | Fluorophore-1 | CTC TTG GGG ACG CTC GAA | Quencher-1 | WT Probe | ||
28839 | Fluorophore-2 | CGC GAT CCC GTC GTT TTA C | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
11358 | 0.40 uM |
28836 | 0.40 uM |
28837 | 0.40 uM |
Wt Probe | 0.15 uM |
Mutant Probe | 0.15 uM |
DNA |
Step | Temp °C | Time | Note |
---|---|---|---|
1 | 94.0 | -- | |
2 | 94.0 | -- | |
3 | 65.0 | -- | -0.5 C per cycle decrease |
4 | 68.0 | -- | |
5 | -- | repeat steps 2-4 for 10 cycles (Touchdown) | |
6 | 94.0 | -- | |
7 | 60.0 | -- | |
8 | 72.0 | -- | |
9 | -- | repeat steps 6-8 for 28 cycles | |
10 | 72.0 | -- | |
11 | 10.0 | -- | hold |