Sequence and conditions of oligos used for PCR amplification and EMSA
Primer Name | Sequence (5′-3′) | Conditions | |
---|---|---|---|
Oligos for amplifying the cDNA | |||
CAR-F1 | GGAGAGGCATTCCATACCAG | 94°C × 30 s, 58°C × 30 s, 72°C × 90 s, 32 cycles | |
CAR-R1 | TTCCCACTCCAGTGTATCCAG | ||
CAR-F2 | CAGGTGACATGCTGCCTAAG | 94°C × 30 s, 57.3°C × 30 s, 72°C × 90 s, 15 cycles | |
CAR-R2 | TATCCAGGGTGTTCCAGGTG | ||
CAR-Taq-F | CCAGCTCATCTGTTCATCCA | 94°C × 30 s, 57°C × 30 s, 72°C × 30 s, 30 cycles | |
CAR-Taq-R | GGTAACTCCAGGTCGGTCAG | ||
CYP2B6-F | CACTCATCAGCTCTGTATTCG | 94°C × 30 s, 57°C × 30 s, 72°C × 30 s, 30 cycles | |
CYP2B6-R | GTAGACTCTCTCTGCAACATGAG | ||
MRP4-F | GCAAGATGCTGCCCGTGTAC | 94°C × 30 s, 60°C × 30 s, 72°C × 30 s, 30 cycles | |
MRP4-R | ACCCTTGCAACTCCTCTCCAAG | ||
Oligos for EMSA | |||
CYP2B6-NR1 | GATCCTGTACTTTCCTGACCCTGAGATC | ||
CYP2B6-NR2 | GATCCATGGACTTTCCTGAACCAAGATC | ||
Oligos used for creating ATG mutants | |||
CAR-ATG-exon1-220-F | GTGGCCTGCAGGTGACTTGCTGCCTAAGAGAAGC* | 95°C × 30 s, 55°C × 30 s, 68°C × 12 min, 12 cycles | |
CAR-ATG-exon-220-R | GCTTCTCTTAGGCAGCAAGTCACCTGCAGGCCAC* | ||
CAR-ATG-exon4-645-F | CCCACACCCGCCACTTGGGCACCATGTTT* | 95°C × 30 s, 55°C × 30 s, 68°C × 12 min, 12 cycles | |
CAR-ATG-exon4-645-R | AAACATGGTGCCCAAGTGGCGGGTGTGGG* | ||
CAR-ATG-exon-654-F | GCCACATGGGCACCTTGTTTGAACAGTTTGTG* | 95°C × 30 s, 55°C × 30 s, 68°C × 12 min, 12 cycles | |
CAR-ATG-exon-654-R | CACAAACTGTTCAAACAAGGTGCCCATGTGGC* | ||
Primers used to amplify the cDNA from hepatocytes | |||
CYP3A4-F | CCAAGCTATGCTCTTCACCG | 95°C × 30 s, 50°C × 30 s, 72°C × 30 s, 28 cycles | |
CYP3A4-R | TCAGGCTCCACTTACGGTGC | ||
CYP2B6-F | TCCTTTCTGAGGTTCCGAGA | 95°C × 30 s, 58°C × 30 s, 72°C × 30 s, 35 cycles | |
CYP2B6-R | TCCCGAAGTCCCTCATAGTG | ||
PXR-F | CAAGCGGAAGAAAAGTGAACG | 95°C × 30 s, 60°C × 30 s, 72°C × 30 s, 28 cycles | |
PXR-R | CTGGTCCTCGATGGGCAAGTC | ||
GAPDH-F | ACCACAGTCCATGCCATCAC | 95°C × 30 s, 60°C × 30 s, 72°C × 30 s, 25 cycles | |
GAPDH-R | TCCACCACCCTGTTGCTGTA |
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↵* Bolded nucleotide indicates the base mutated to change ATG to TTG.