The comparison of both primer systems was conducted based on all available genera described in the list of prokaryotic names in the nomenclature (http://www.bacterio.cict.fr/index.html).

Subsequently, a simple matching coefficient (Jaccard coefficient) was calculated as described below: To revise the in vitro primer specificity, 16S RNA gene fragments were learn more amplified by Com2xf/Ac1186r-PCR, using DNA from plaster and compost materials and both bioaerosol samples. The genomic DNA from the 18 different building material samples was used to verify the new primer system Com2xf/Ac1186r for screening of 16S rRNA gene clone libraries, generated using 27f-1492r primers (Lane, 1991). For screening of these clone libraries, the Actinobacteria-specific primer system www.selleckchem.com/products/Etopophos.html developed by Stach et al. (2003) was used to compare the detectable Actinobacteria species. All PCR amplification reactions were performed in a final volume of 25 μL, containing 10 mM Taq buffer (+KCl), each primer at 200 nM, each dNTP at 0.2 mM, 2.0 mM MgCl2 and 0.02–0.025 U of Taq. Primers, cycling parameters and concentrations for PCR contents are shown in Tables 2 and 3. Amplification programs were started by an initial denaturation step at 95 °C for 3 min

and finished with a final extension step at 72 °C for 15 min and 30 min for add-on cloning analyses. Reactions were performed in a Thermocycler (My Cycler™, BioRad, Munich, Germany). A control PCR using only PCR reagents was always carried out. PCR comprised 25 cycles, except primer system 27f/1492r (35 cycles). PCR reactions contained 4 mg mL−1 bovine serum albumin. Negative PCR controls containing nuclease-free water instead of DNA were always carried out. check The PCR products were visually evaluated by 1% agarose gel electrophoresis with ethidium bromide staining. PCR was purified using a QIAquick PCR Purification Kit (Qiagen, Hilden, Germany) and quantified photometrically (Ultrospec 4000, Amersham Biosciences, Freiburg, Germany).

Cloning analyses from four environmental samples (plaster, mature compost and two bioaerosols) and subsequent sequencing of clone inserts was done by Agowa (Berlin, Germany) using the M13F primer (Invitrogen Corp., Carlsbad, CA). For construction of 16S rRNA gene clone libraries from building material samples, the cloning kit Promega p GEM-T® Vector Systems (Madison, WI) was used with 27f-1492r primers according to the manufacturer’s instructions. Here, 100 white colonies from each sample were picked and incubated overnight at 37 °C on Luria–Bertani (LB) agar containing ampicillin (100 μg mL−1), X-Gal (80 μg mL−1) and IPTG (100 mM) (Sambrook & Russel, 2001). Clone inserts were reamplified to screen all generated clones for affiliation to Actinobacteria with the Actinobacteria-specific primer systems (Com2xf/Ac1186r SC-Act-235aS20/SC-Act-878aA19).