ARK-Genomics, Roslin Institute, Roslin, Midlothian, EH25 9PS, United Kingdom
Manufacture protocol
The clone-set was amplified in 96 well plates and re-arrayed into a 384-well Montage (Millipore) filtration plate to isolate the amplified DNA products. These products were checked by agarose gel electrophoresis and quantified using a Picogreen DNA quantitation assay. All single band PCR products were re-arrayed, in fresh barcoded 384 well pates (Genetix 7022), at a concentration of 150ng per microlitre in 0.1M sodium phosphate buffer pH 8 containing 0.01% SDS, ready for printing. All re-arraying was done with Multiprobe II (Perkin Elmer) liquid handling robot. DNA was printed onto aminosilane coated glass slides (Corning GAPS II) using a Genomic Solutions MicroGrid II arrayer and the MicroSpot pins according to the manufacturers instructions. All printing was carried out in conditions of constant humidity (52% RH) and temperature (20oC) in a room supplied with HEPA filtered air. Prior to printing the pins were cleaned by ultra-sonication in water and if necessary 50mM potassium hydroxide. A test print of all pins was carried out by printing Cy3 streptavidin conjugate dye in printing buffer onto poly lysine slides and printing 100 features per pin. When all pins were printing consistent size spots for more than 80 features they were accepted as ready to print. The MicroGrid II was then programmed for the array print run, with a grid layout file prepared to allow duplicate printing at random within a patch. At the end of the print run the slides were removed following the program instructions in preparation for post processing. The slides are processed to bind the DNA to the slide, denature the DNA, and prevent non-specific binding during hybridisation. Slides were transferred from the printer into stainless steel slide staining racks before baking at 80oC for 4 hours in an oven. The slides were blocked after they had returned to room temperature. Blocking was carried out in a fume hood. Once in solution, the blocking agents do not remain active for long, so it is important to prepare fresh solutions and use them immediately. Dissolve 0.75g succinic anhydride completely in 125ml 1-methyl-2-pyrrolidinone. Immediately mix in 125ml of 0.2M boric acid pH 8.0. The slides were plunged into the solution and vigorously agitated for 15 minutes using a magnetic stirrer. The slides were then transferred to a stirred water bath containing MilliQ water maintained at 95 oC and plunged up and down and side to side for 2 minutes to denature the DNA to single strands. The slides were then transferred to a trough containing 96% ethanol for 1 minute. The slides were then transferred to microscope slide boxes and dried by centrifugation. Slides were then stored in a desiccator in the dark at room temperature until use.
Support
glass
Coating
aminosilane
Description
This is a chicken multi-tissue cDNA microarray with 12752 elements. It comprises clones from chicken EST collections generated by BBSRC, University of Delaware and the Fred Hutchinson Cancer Research Center. Sequence information is available from GenBank for all features on the array.
