Preparation of the iron chip microarray platform Clone amplification and purification: 894 murine 'expressed sequence tag' (EST) clones,that were sequence verified from both ends were chosen for the iron chip. The ESTs were selected to contain the 3'end of a cDNA (i.e. the polyadenylation signal) and to extend for at least 300bp towards the 5'end. In addition,15 positive and negative controls were chosen,that are derived from bacterial and arabidopsis genomes (see below). All clones were PCR amplified with 5´amino-alkyl modified vector primers using the Roche Diagnostics Expand High Fidelity PCR System. The PCR products were purified on a BIOMEK 2000 robot (Beckman),using the Macherey-Nagel NucleoSpin Multi-96 Extract PCR purification system. After purification,5 µl of the purified PCR reaction were subjected to quality control on 1% agarose gels. Size,quality (single vs. multiple bands) and concentration of the PCR fragments were determined. SSC buffer was added,resulting in a final concentration of 2xSSC. 9 µl of the diluted PCR products were transferred to a 384-well plate for spotting. Spotting and attachment: Silanised glass slides were used as a platform for spotting on the Omnigrid (GeneMachines) spotting robot. Split pins from Telechem (spot size 120 µm) were used. All clones were spotted in triplicates next to each other. External controls and selected house keeping genes were spotted in dilutions 1:1,1:2 and 1:4. Cy3 labelled oligonucleotides were spotted to control the performance of the spotting needles and DNA attachment quality in four of the eight resulting subgrids. Spotting was performed at 24°C,60% relative humidity. After spotting,the slides were immediately placed in a 50°C humid oven for 3 hours and additionally baked at 100°C for 10 min,to stabilise DNA binding to the silanised glass surface
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