Methods Strains and culture conditions Nostoc punctiforme ATCC 29133 cultures were grown in CYT387 purchase BG110 medium [40] either in 100 ml Erlenmeyer flasks on a shaking table or on plates containing BG110 medium solidified by 1% noble agar (Difco). Larger volumes of N. punctiforme cultures were grown in 1 L Erlenmeyer
flask containing BG110 medium under continuous stirring and sparging with air. All cultures were grown at 25°C at a continuous irradiance of 40 μmol of photons m-2 s-1 (29). For cultures treated by sonication or were electroporated, the BG110 medium was supplemented with 5 mM MOPS (pH 7.8) and 5 mM NH4Cl as Selleck INCB28060 a combined nitrogen source. 10 μg/ml ampicillin
was used for selection of positive clones after electroporation with the vector constructs. All cloning was done using Escherichia coli strain DH5α grown at 37°C in Luria broth (LB) liquid medium [41], supplemented with 100 μg/ml ampicillin, and on plates containing LB medium solidified with 1% agar and supplemented with 100 μg/ml ampicillin. PCR, DNA sequencing and sequence analysis Genomic DNA was isolated from N. punctiforme cultures as previously described [12]. The concentration was determined by absorbance measurements using Cary Win UV (Varian). PCR amplifications were carried out using the high fidelity DNA polymerase Phusion (Finnzymes), according to manufacturer’s protocol, in a GeneAmp PCR system 2400 (Applied Biosystem). The primers used in this pheromone work are listed in Table 1. All primers were designed
using the Primer3 program http://frodo.wi.mit.edu/cgi-bin/primer3/primer3_www.cgi and blasted against the N. punctiforme genome [42] (JGI Microbial genomes, http://genome.jgi-psf.org/mic_home.html), or in the case of sequencing primers against their CB-839 price corresponding vector sequence (Table 1), to check their specificity. Secondary structure of the primers was analysed with the Primer design utility program http://www.cybergene.se/primerdesign/. Amplified DNA fragments were isolated from agarose gels using the GFX PCR DNA and Gel Band Purification Kit (GE Healthcare), following the manufacturer’s instructions. Sequencing reactions were performed by Macrogen Inc. and computer-assisted sequence analyses were performed using BioEdit Sequence Alignment Editor Version 7.0.5.3.