Accuprep plasmid miniprep dna extraction kit

Restriction enzymes were purchased from New England Biolabs; T4 DNA ligase and Taq DNA polymerase were from Invitrogen; Velocity proof-reading DNA polymerase was from Bioline; Q5 high fidelity Taq polymerase was from NEB. PCR amplification of DNA was achieved using the primers (AltaBioscience, University of Birmingham, UK; or Sigma Aldrich) listed in S3 Table. Reactions were cycled in a SensoQuest Lab Cycler following standard procedures [57 ]. PCR products were purified using the Illustra GFX^TM PCR DNA and Gel Band Purification Kit (GE^TM Healthcare). Small-scale plasmid DNA preparations were performed using the AccuPrep Plasmid MiniPrep DNA Extraction Kit (Bioneer) adapted from the alkaline lysis method of Birnboim and Doly [58 (link)]. DNA sequencing reactions were prepared and run on an ABI 3730 DNA analyser (Functional Genomics Facility, University of Birmingham, U.K.) following the chain termination method [59 ].

To evaluate an association between changes in the chromosomal carbapenemase gene sequence of isolates and their antimicrobial resistance pattern, a two-step approach was adopted. An initial AFLP assay was carried out on bla_{OXA-23-like⁺} CR-AB, followed by DNA sequence analysis of the bla_OXA-23-like gene of a representative isolate from each AFLP genotype group.
Briefly, we used a high fidelity Pfu DNA polymerase (Fermentas, Lithuania) to generate bla_OXA-23-like specific amplicons, which were purified using a AccuPrep® PCR Purification Kit (Bioneer, Daejeon, Korea) and cloned into pTZ57R (InsT/A Clone PCR product cloning kit, Fermentas, Vilnius, Lithuania). DNA was then transferred into competent E. coli TOP10 cells, which were then isolated using Luria-Bertani (LB) agar supplemented with ampicillin (100 μg/mL). Plasmid DNA was prepared with the AccuPrep Plasmid MiniPrep DNA Extraction Kit, (Bioneer, Daejeon, Korea) and sequenced using an ABI3730 automatic sequencer (Applied Biosystems, CA, USA). The sequences were analyzed using a BLAST algorithm against the NCBI GenBank database [http://www.ncbi.nlm.nih.gov/guide/dna-rna/ (accessed 05.06.11)].

Escherichia coli DH5α and BL21 (DE3) strains were used as the cloning and expression hosts, respectively. Both strains were grown in Luria–Bertani broth (LB broth) at 37 °C with agitation at 180 rpm. The pET-16b (Novagen, Madison, USA) vector was used for enzyme expression and purification. Buffers and enzymes used for the polymerase chain reaction (PCR) and DNA manipulation were purchased from Takara (Takara Bio Inc., Shiga, Japan). PCR products were purified using an AccuPrep^® Gel Purification Kit (Bioneer, Daejeon, South Korea), and cloned plasmids were extracted using an AccuPrep^® Plasmid MiniPrep DNA Extraction Kit (Bioneer). Substrates and other reagents used for the enzyme assay, including p-nitrophenyl acetate (p-NPA), p-nitrophenol (p-NP), d-xylose, and dinitrosalicylic acid, were purchased from Sigma-Aldrich (St Louis, MO, USA). The synergistic effect was verified with commercially available endo-1,4-β-xylanase derived from Aspergillus niger (Megazyme Int., Wicklow, Ireland) using beechwood xylan (Tokyo Chemical Industry Co. Ltd., Tokyo, Japan) as the substrate.