title: MH Hannover Pseudomonas putida 4.5K
status: Public on Jul 27 2006
contact.city: Hannover
contact.first: Burkhard
contact.last: Tummler
contact.email: tuemmler.burkhard@mh-hannover.de
contact.address: Carl-Neuberg-Str. 1
contact.department: OE 6710
contact.country: Germany
contact.institute: Medizinische Hochschule Hannover
contact.phone: +49-511-5322920
organism: Pseudomonas putida
manufacture_protocol: Purified DNA of some 4600 overlapping shotgun clones in vector pCR-BluntII-TOPO (Invitrogen, Groningen, The Netherlands) was used as template in standard PCR amplification with the vector-specific primer pair d(TCGGATC CACTAGTAACG) and d(GGCCGCCAGTGTGATG). Shotgun cloning had been performed according to the procedures described by the vector's manufacturer. Each PCR reaction was individually checked by agarose gel electrophoresis. Prior to spotting onto microarrays, the DNA concentration of the fragments was adjusted to about 300 ng μl1. Spotting was done using an SDDC-2 DNA Micro-Arrayer from Engineering Services Inc. (Toronto, Canada) and SMP3 pins (TeleChem International Inc., Sunnyvale, USA) onto glass slides coated with poly L-lysine as described in detail earlier (Diehl et al., 2001).
  For labelling DNA-samples, random priming (Feinberg and Vogelstein, 1983) was performed on 500 ng genomic Pseudomonas DNA in the presence of 15 μM each of dATP, dGTP and dTTP, and 25 μM Cy3- or Cy5-labelled dCTP (Amersham-Pharmacia, Amersham, UK). Hybridization of such samples to the microarrays was done as described (Diehl et al., 2001). Fluorescence signals were detected on a ScanArray5000 unit (Packard, Billerica, USA) and analysed with the GenePix software package (Axon Instruments, Union City, USA).