Abi prism 373 genetic analyzer

The sequences of the miR-497 precursor (MIR497) and the miR-34a precursor (MIR34A) were amplified with oligonucleotide primers synthesized by Invitrogen (Figure S4). MIR497 and MIR34A were then inserted individually into the pcDNA™6.2-GW/EmGFPmiR vector (Invitrogen) using the BLOCK-iT™ Pol II miR RNAi Expression Vector Kit (Invitrogen), to generate the expression constructs Hi-miR497 and Hi-miR34a, respectively. These were verified by DNA sequencing with the primer 5′- CTCTAGATCAACCACTTTGT-3′ in an ABI Prism 373 Genetic Analyzer (Applied Biosystems). The empty pcDNA™6.2-GW/EmGFPmiR vector was used as the negative control (mock). To investigate the cooperative activities of miR-497 and miR-34a, we used the isocaudomers BamHI (NEB, Beverly, MA) and BglII (NEB) to ligate both MIR497 and MIR34A into the vector to generate the expression construct Hi-miR497/34a, which was confirmed by DNA sequencing.
A clone of the green fluorescent protein (GFP)-tagged cDNA [including complete 3-UTR encoding human CCNE1 (NM_001238.2)], was bought from Origene (Rockville, MD) as transfection-ready plasmid DNA. The neomycin-resistance gene is expressed downstream from the SV40 promoter in the same vector, which permits the positive selection of transfected cells. To construct cells stably expressing CCNE1, A549 cells were transfected with the plasmid DNA and selected with neomycin (0.5 mg/mL).

Multiplex PCR to detect ESBL or PABL genes was performed as described previously [1 (link)]. DNA fragments were separated on a 1% agarose gel. Fragments of the appropriate size [1 (link)] were extracted from the gel and purified using a Gel Extraction kit (Qiagen Inc., CA, USA), followed by sequencing in an ABI Prism 373 Genetic Analyzer (Applied Biosystems, Foster City, USA) using Sanger’s method. A database search was then performed using the BLAST program at NCBI (http://www.ncbi.nlm.nih.gov).