5 mM MgCl2, 2 5 μL dimethyl sulfoxide, 5 μL of 10 × PCR buffer [1

5 mM MgCl2, 2.5 μL dimethyl sulfoxide, 5 μL of 10 × PCR Temsirolimus in vivo buffer [100 mM Tris-HCl (pH 8.3),

100 mM KCl] and 2.5 units of Taq DNA polymerase (Fermentas, Hanover, MD, USA), and adding ddH2O to a final volume of 50 μL. The PCR program consisted of an initial 5 min denaturation step at 94°C; 30 cycles of 1 min at 94°C, 1 min at 50°C, 1.5 min at 72°C; and a final extension step at 72°C for 5 min. Table 1 Primers used in this study Primer Sequence Reference Uni-27F 5′-AGAGTTTGATCMTGGCTCAG-3′   Uni-1492R 5′-GGYTACCTTGTTACGACTT-3′ 49 Primers #1F 5′-GTSGGBTGYGGMTAYCABGYCTA-3′   Primers #1R 5′-TTGTASGCBGGNCGRTTRTGRAT-3′ 15 darsB1F 5′-GGTGTGGAACATCGTCTGGAAYGCNAC-3′   darsB1R 5′-CAGGCCGTACACCACCAGRTACATNCC-3′ selleck chemicals 16 dacr1F 5′-GCCATCGGCCTGATCGTNATGATGTAYCC-3′   dacr1R 5′-CGGCGATGGCCAGCTCYAAYTTYTT-3′ 16 dacr5F 5′-TGATCTGGGTCATGATCTTCCCVATGMTGVT-3′   dacr4R 5′-CGGCCACGGCCAGYTCRAARAARTT-3′ 16 B = G, T or C; M = A or C; N = A, C, G, or T; R = A or G; S = G or C; V = A, C, or G; Y = C or T. Colony morphologies and 16S rDNA PCR-RFLP technique were used to remove the repeated isolates for each sample. PCR-RFLP was performed by enzyme digestion at 37°C for 3 hrs in a 20 μL volume containing 2 μL of 10 × enzyme buffer, 2.5 units of HaeIII or MspI and 5–10 μL of the 16S rDNA PCR products, amending ddH2O to a final volume of 20 μL. The digested DNA fragments were

separated in 2% agarose gels and the digestion patterns were grouped by DNA fingerprinting profiles. Identification of the

aoxB gene encoding the arsenite oxidase Mo-pterin subunit and arsB, ACR3(1) and ACR3(2) genes encoding different arsenite transport proteins The PCR amplification of aoxB was performed Crenolanib research buy using degenerate primers (Primers #1F and #1R) (Table 1) and following the PCR conditions as described by Inskeep et al. [15]. The check details amplification of arsB, ACR3(1) and ACR3(2) genes were performed using three pairs of degenerate primers [darsB1F and darsB1R for arsB, dacr1F and dacr1R for ACR3(1), dacr5F and dacr4R for ACR3(2)] (Table 1) as described by Achour et al. [16]. The PCR products were purified using the Gel Extraction Kit (SBS Genetech, Shanghai, China). The purified PCR products were ligated into pGEM-T (Promega, Madison, WI, USA) and the ligation products were used to transform E. coli DH5α competent cells by electroporation. The transformants were grown on LB agar containing ampicillin, X-Gal and IPTG at 37°C for 16 hrs according to the manufacturer’s recommendations. DNA sequencing and phylogenetic analysis The PCR products were purified using the UltraPure™ PCR Kit (SBS Genetech). DNA sequencing analysis was performed using ABI 3730XL DNA analyzer by Sunbiotech company (Beijing, China). All sequences were analyzed by BlastN (for 16S rRNA gene) and BlastX (for deduced AoxB and ArsB/Acr3p) searching tools [50]. All sequences were checked manually and edited for the same lengths using ClustalX 1.83 software [51]. MEGA 3.

Leave a Reply

Your email address will not be published. Required fields are marked *

*

You may use these HTML tags and attributes: <a href="" title=""> <abbr title=""> <acronym title=""> <b> <blockquote cite=""> <cite> <code> <del datetime=""> <em> <i> <q cite=""> <strike> <strong>