Directly after C4 a reed node followed C1 and C4 were thus the n

Directly after C4 a reed node followed. C1 and C4 were thus the newest and oldest chamber harboring most sellckchem likely a female and a male, respectively, and were treated separately as two individual samples in the following. Chambers were not visibly affected by macro-pathogens, parasites or nest destroyers. As far as observable, they were intact and the individuals were healthy. DNA extraction For each chamber, we sampled all nest contents combined (including bees, cocoon, remaining pollen) for DNA extraction. Further swabs of the complete interior i.e. chamber walls and loam barriers were taken with sterile cutton buds. The merged pools of chamber contents were extracted with a spatula and together with the swabs transferred into the kit lysis buffer.

We used the MoBio PowerSoil DNA Isolation kit (Carlsbad, CA, USA), adequate for microbial DNA extraction in environmental samples. The substance in the buffer was mechanically disrupted with an electric hand-held homogenizer. The following extraction and isolation steps were performed according to the manufacturers instructions. The extraction kit uses a silica bead-beating step, which was performed on a vortexer with self-made horizontal probe mounts for 10 minutes at maximum speed. All tasks were performed with gloves and our tools were sterilized with 70% ethanol between sampling steps. Amplicons For PCR amplification, we used primers to amplify 16S ribsomal DNA of bacteria according to Hamady et al. [31] that enclose the variable regions V1-V2. It was found to be well suited for the phylogenetic analysis of pyrosequencing reads [31-34].

We used ��fusion�� primers designed to have 454 adapter regions and the targeting primers. The forward ��fusion�� primer (5��-CGTATCGCCTCCCTCGCGCCA-TCAG-AGAGTTTGATCCTGGCTCAG-3��) consists of the 454 specific Adapter A, the linker key and the conserved forward primer 27f (the corresponding regions are delineated in the sequence by hyphens). The reverse ��fusion�� primer (5��-CTATGCGCCTTGCCAGCCCGC-TCAG-XXXXXXXXXX-CATGCTGCCTCCCGTAGGAGT-3��) contains the 454 specific Adapter B, the linker key, an multiplex identifier (MID) and the bacterial primer 338f (regions delineated in the sequence by hyphens). MIDs were used to analyze several samples together on the same sequencing chip. For our two nest samples we used the MIDs 5 (ATCAGACACG) and 7 (CGTGTCTCTA) officially suggested by Roche in a technical bulletin (454 Sequencing Technical Bulletin No.

005-2009, April 2009). Their position is indicated by placeholders ��X�� in the aforementioned reverse ��fusion�� primer sequence. The remaining MIDs were used for different projects using the same primers and target organisms (bacteria). Fusion primers were constructed at the Metabion laboratories (Martinsried, Germany). PCR reaction mixes consisted GSK-3 of 0.

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