title: ILLING Xerophyta 1709 v1.0.
status: Public on Mar 30 2012
contact.city: Rondebosch
contact.first: Nicola
contact.last: Illing
contact.email: nicola.illing@uct.ac.za
contact.state: Western Cape
contact.address: Chemistry Rd
contact.department: Molecular and Cell Biology
contact.country: South Africa
contact.institute: University of Cape Town
contact.phone: 27216502414
organism: Xerophyta humilis
manufacture_protocol: (1) The inserts of 3105 X. humilis clones (1450 clones from LD cDNA library;  1453 clones from RD cDNA library; 107 clones from LR cDNA library; and 95 clones from RR cDNA library) were PCR amplified in Corning Thermowell 96-well plates (Corning Incorporated, NY, USA).  For each cDNA clone, 1 µl of 25% glycerol stock culture was added to 100 µl of a PCR mixture containing 0.3 µM T3 primer, 0.3 µM T7 primer, 200 µM dNTPs, 2.5 mM MgCl2, 1X PCR reaction buffer and 0.1 µl (5U/µl) Super-therm polymerase (Southern Cross Biotech, South Africa). PCR was performed as follows: at 94°C for 1 min; for 10 cycles at 94°C for 20 sec, 56°C for 20 sec, 72°C for 2 min; for 30 cycles at 94°C for 20 sec, 51°C for 20 sec, 72°C for 2 min; and at 72°C for 7 min.
  (2) PCR reactions were transferred to Millipore 96-well Multiscreen PCR plates (Millipore, MA, USA) fit on vacuum apparatus, then allowed to pass through the filters for 10 minutes.  Filters were then washed by passing 100 µl H2O for 10 minutes twice.  PCR products were resuspended in 50 µl 50% DMSO with vigorous agitation for 60 minutes. Twenty microlitres of which were pipette-transferred into 384-well plates (Amersham, Germany) for slide printing.
  (3) The purified cDNAs, together with Lucidea controls (Amersham Biosciences, Germany) were printed onto Corning GAPS II coated slides (Corning Incorporated, NY, USA) by MicroGrid II Arrayer (BioRobotics, Cambridge, UK) with 16 10 µm split pins in a 4x8 configuration following manufacturer's instructions. The printed slides were UV-crosslinked (900 mJ cm2), and baked at 80°C for 2 hours. Each printed block was designed to contain random unbiased mixture of cDNAs originated from LD, RD, LR and RR library clones.