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Additional info for Diagnostic Bacteriology Protocols 2nd Edition (Methods in Molecular Biology Vol 345)
Spotting of Glass Slides 1. Spot the slides using an Affymetrix 417 Arrayer equipped with 125 µm pins, which results in spots approx 200 µm diameter. 2. Before and during spotting, ambient temperature and air humidity must be controlled and maintained at 20ЊC and 50 to 60% relative humidity, respectively (see Note 7). 3. The spotting layout (see Note 8) is determined by the print head geometry and the well positions of specific DNA oligos within the plates. Each slide can easily hold two arrays, which require a 22 ϫ 22-mm area for on-chip PCR.
McGregor, D. , et al. (1996) Simultaneous detection of microorganisms in soil suspension based on PCR amplification of bacterial 16S rRNA fragments. BioTechniques 21, 463–470. 17. Iqbal, S. , Mayo, M. , Bruno, J. , Bronk, B. , Batt, C. , and Chambers, J. P. (2000) A review of molecular recognition technologies for detection of biological threat agents. Biosens. Bioelectron. 15, 549–578. 18. Alvarez, A. , Buttner, M. , and Stetzenbach, L. D. (1995) PCR for bioaerosol monitoring: sensitivity and environmental interference.
Glass cover slips (22 ϫ 22 mm), for microarray applications. 8. PTC 200 in situ slidecycler (MJ Research [Bio-Rad]). 9. 4 (as required during DNA extraction). 5. Fluorescence Staining and Scanning 1. 0. Store as 20X stock solution. 2. 10% (w/v) SDS solution. 3. Streptavidin-Alexa Fluor 647 conjugate (Molecular Probes). Store under light protection according to the manufacturer’s instructions. 4. 0. 5. Affymetrix 428 laser scanner (Affymetrix), or similar microarray scanner. 6. GenePix software (Axon Instruments), or any other software for microarray analysis.