Ab13100 dna sequencer

PCR was performed with HotStar Taq polymerase (Qiagen) according to the manufacturer's instructions and specific oligonucleotide primers for detection of the following genes: mecA, mecC, SCCmec, agr locus (agr-1_Sa to agr-4_Sa), lukE-lukD, lukS-PV, and lukF-PV, norA, qacA/B, qacC/qacD, qacG, qacH, blaZ, mupA, vanA, aac6′/aphD (Oligonucleotide primers are listed in Supplement 1). Amplified PCR products were purified with Qiagen purification kit (Qiagen Valencia, CA, USA) according to the manufacturer's instructions and both strands were sequenced by automated AB13100 DNA sequencer (Applied Biosystems, Carlsbad, CA, USA) system. The BLAST program of the National Centre for Biotechnology Information (http://www.ncbi.nlm.nih.gov) was used to search and compare databases for similar nucleic acid sequences.

Colony PCRs were performed to detect the presence of four main class D OXA β-lactamase genes (bla_{OXA–23-like}, bla_{OXA–24-like}, bla_{OXA–51-like}, and bla_{OXA–58-like}) and three class B, also known as metallo-β-lactamases, genes (bla_IMP, bla_VIM, and bla_SIM). Isolates confirmed positive for oxacillinases genes were further screened for the presence of ISAba1 element upstream of these genes [27 (link)]. Briefly, colony PCRs were performed in a 50 μl reaction mixture with 25 μl of EconoTaq PLUS GREEN 2X Master Mix (Lucigen, Middleton, WI), 0.5 μl each of forward primer (100 μM) and reverse primer (100 μM), and 25 μl of nuclease-free water. Small amount of bacterial colony was removed using the sterile toothpick and added to the master mix. Amplification was carried out under the following conditions: denaturation at 94°C (3 min), 35 cycles of denaturation at 94°C (45 s), annealing for 45 s as described previously [28 (link)], and extension at 72°C for 1 min, followed by a final extension of 72°C for 1 min. The Qiagen purification kit (Qiagen, USA) was used to purify amplified PCR products, and both strands were sequenced by automated AB13100 DNA sequencer (Applied Biosystems) system. The nucleotide sequences were analyzed using the basic local alignment search tool (BLAST).

Coding sequences of tax and p53 genes were analyzed using AB13100 DNA sequencer (Applied BioSystems).