PMC:13912 / 16110-17971 JSONTXT

{"target":"https://pubannotation.org/docs/sourcedb/PMC/sourceid/13912","sourcedb":"PMC","sourceid":"13912","source_url":"http://www.ncbi.nlm.nih.gov/pmc/13912","text":"Polymerase chain reaction\nTwo different primer sets, ERD3 and ERD5, were used to coamplify \t\t\t WT-ER and ERD3 complementary DNAs, and WT-ER and ERD5 complementary DNAs, \t\t\t respectively. ERD3 primer set consisted of D3U primer (5' \t\t\t -TGTGCAATGACTATGCTTCA-3', sense, located in WT-ER exon 2, position \t\t\t 792-811) and D3L primer (5' -TGTTCTTCTTAGAGCGTTTGA-3', antisense, \t\t\t located in WT-ER exon 4, position 1145-1125). ERD5 primer set consisted of D5U \t\t\t primer (5' -CAGGGGTGAAGTGGGGTCTGCTG-3', sense, located in WT-ER \t\t\t exon 4, position 1060-1082) and D5L primer (5'-α \t\t\t TGCGGAACCGAGATGATGTAGC-3', anti-sense, located in WT-ER exon 6, position \t\t\t 1542-1520). The given positions correspond to published sequences for WT-ER \t\t\t [1].\nPCR amplifications were performed and PCR products analyzed as \t\t\t previously described [18]. Briefly, 0.2 μ l reverse \t\t\t transcription mixture was amplified in a final volume of 15 μ l, in the \t\t\t presence of 1.5 μ Ci of [α-32P] dCTP (3000 Ci/mmol), \t\t\t 4ng/μ l of each primer of the primer set considered (ERD3 or ERD5 primer \t\t\t set) and 0.3 unit of Taq DNA polymerase. Each cycle consisted of 30s at \t\t\t 94°C, 30s at 60°C and 30s at 72°C. PCR products were then \t\t\t separated on 6% polyacrylamide gels containing 7mol/l urea (polyacrylamide gel \t\t\t electrophoresis). Following electrophoresis, the gels were dried and \t\t\t autoradiographed. For each PCR, two PCR products were obtained, which were \t\t\t identified by subcloning and sequencing. PCR products migrating with the \t\t\t apparent size of 354 and 483 base pairs, using ERD3 and ERD5 primer set, \t\t\t respectively, were shown to correspond to WT-ER complementary DNA. PCR products \t\t\t migrating with the apparent size of 237 and 344 base pairs, using ERD3 and ERD5 \t\t\t primer set, were shown to correspond to ERD3 and ERD5 complementary DNAs, \t\t\t respectively.","divisions":[{"label":"Title","span":{"begin":0,"end":25}}],"tracks":[]}