title: Univ Wuerzburg Blochmannia floridanus 564 v1.0
status: Public on Nov 10 2008
contact.city: Wuerzburg
contact.first: Sascha
contact.last: Stoll
contact.email: stoll@biozentrum.uni-wuerzburg.de
contact.address: Biozentrum am Hubland
contact.department: Dpt. of Microbiology
contact.country: Germany
contact.institute: University of Wuerzburg
organism: Candidatus Blochmanniella floridana
description: whole genome DNA macroarray
manufacture_protocol: Gene-specific PCR primer pairs were designed and synthesized by MWG Biotech (Ebersberg, Germany) based on the Bl. floridanus genome sequence (RefSeq accession no NC_005061.1) to yield products of approximately 500 bp. We amplified 564 genes in duplicate 50 µl reactions, omitting the genes for most non-coding RNAs and several ribosomal subunit proteins. Reactions contained 1x PCR buffer with 1.5 mM MgCl2, 0.2 mM dNTPs, 0.8 µM each primer, 1.25 units Taq polymerase (Molzym, Bremen, Germany) and 4 ng of purified Bl. floridanus genomic DNA. Products were checked by agarose gel electrophoresis for correct size and purity. The DNA concentration of each product was measured photometrically and DNA was diluted to 100 ng/µl in sterile distilled water. PCR products were spotted in duplicate (approx. 30 ng DNA per spot) on 7x11 cm nylon filters together with an external control PCR product from a kanamycin resistance gene (neo) as the central spot of each 3x3 spot group (Eickhoff et al., 1999) as well as a PCR reaction without primers and template as a negative control at the German Resource Center for Genome Research (Berlin, Germany).