title: PhotorhabdusTT01-4144 genes
status: Public on Oct 13 2010
contact.city: Montpellier
contact.first: Sophie
contact.last: Gaudriault
contact.email: sgaudriault@univ-montp2.fr
contact.address: case courrier 54, Place Eugène Bataillon
contact.country: France
contact.institute: Université Montpellier 2 - Institut National de la Recherche Agronomique
contact.phone: 33 4 67 14 48 12
organism: Photorhabdus laumondii subsp. laumondii TTO1
description: spotted PCR amplicons (300-600 bp)
manufacture_protocol: 1. Primers were designed by use of a modified version of Primer 3 software (CAAT-Box) to amplify specific fragments (300 to 600 bp).
  2. Primers were synthetized by Operon (25pmol/µl). The mix of the forward and reverse primers were provided in 96 deepwell plates.
  3. Primers were transfered in 384 well plates Thermofast Abgenes (Merck) and mixed  with dNTP (25mM),  Titanium DNA polymerase (5U/µL; Clontech) and Photorhabdus luminescens TT01 genomic DNA (30 ng/µL).
  4. Probes were amplified in a Genmate robot (hybridization temperature: 62°C, elongation time: 1 min; 35 cycles). Two rounds of PCR were performed in order to have more than 1 µg of PCR product.
  5. Quality and quantity of the final matrix were checked by gel electrophoresis of the amplified probes and sequencing of 96 randomly chosen amplified probes.
  6. Probes wer purified on Multiscreen PCR filter plates according to the manufacturer protocol (Millipore).
  7. The probes concentration were evaluated with picrogreen labelling and spectrophotometer measure (Spectra Fluor S).
  8. The plates were lyophilized at low temperature and the probe concentrations were adjusted to 30ng/µl in 50% DMSO.
  9. 10 µL of each well was transferrred in a 384 well plate compatible with the spotter Gen III (Amersham).
  10. Spotting was performed using the Gen III Amersham spotter (distance between spots : 260 µm, spots diameter :230 µm, atmosphere humidity : 50%). Each probes is spotted twice on a slide (Rep1 and Rep2 in the raw data files)