title: Pm 5K Random Array II
status: Public on Nov 21 2013
contact.city: University of Melbourne
contact.first: Alex
contact.last: Andrianopoulos
contact.email: alex.a@unimelb.edu.au
contact.state: Victoria
contact.address: Royal Parade
contact.department: Department of Genetics
contact.country: Australia
contact.institute: University of Melbourne
organism: Talaromyces marneffei
manufacture_protocol: 1. Two small insert (400-600 bp) DNA libraries of partially digested P. marneffei genomic DNA were generated using Sau3AI or TaqI cloned into the BamHI or ClaI sites of pALX223, respectively.
  2. Independent colonies from both libraries were selected for the construction of the microarrays
  3. Colonies (5376) were picked using the BioRobotics BioPick machine and individually placed into 384 well plates containing LB broth.
  4. Colonies were then grown overnight, DNA prepared from a 2 ml aliquot of each culture and used to PCR amplify the gDNA inserts with M13 forward and reverse primers.
  5. The resulting PCR products were purified using Millipore Multiscreen filter plates via a manifold vacuum. The quality and quantity of purified amplified product were ascertained through agarose gel electrophoresis.
  6. Amplified DNA inserts were adjusted to 250 ng/ml in 50% DMSO buffer for printing.
  7. PCR products from 38 previously characterised P. marneffei sequences were used as controls. Gene IDs for these sequences are PMAA_085540, PMAA_003830, PMAA_006930, PMAA_011430, PMAA_011430, PMAA_014540, PMAA_021220, PMAA_030430, PMAA_030490, PMAA_032950, PMAA_043500, PMAA_054000, PMAA_054000, PMAA_056390, PMAA_056390, PMAA_067280, PMAA_067670, PMAA_067970, PMAA_068110, PMAA_070290, PMAA_071350, PMAA_075300, PMAA_078000, PMAA_078530, PMAA_078530, PMAA_079960, PMAA_080160, PMAA_080470, PMAA_083100, PMAA_084900, PMAA_084960, PMAA_086720, PMAA_093460, PMAA_093460, PMAA_095260, PMAA_099990, PMAA_100040, PMAA_100070.
  8. Scorecard Version 2 were also used as controls.
  9. The amplified DNA inserts were arrayed in duplicate onto Corning GAPS II aminosilane glass slides using the ESI Virtek Arraying Robot. DNA was spotted using Point Technology split pins.
  10. Slides were baked and UV irradiated to crosslink the DNA.