Seccion de Chips de DNA-SCSIE (Universitat de Valencia)
Manufacture protocol
Each selected ORF was amplified with specific primers from genomic DNA of Lactobacillus casei ATCC 334. As a general rule, the primers were selected to amplify the complete coding region excepting ORFs exceeding 2.5 kb for which a 2.5 kb internal fragment was amplified. PCR products were analyzed in agarose gels and their sizes estimated by comparison with standard markers. BioGrid (BioRobotics, UK) was used as the spotting robot. Macroarrays were made by printing the PCR products (without purification) onto a positively charged nylon membrane (Amersham Hybond N+). Printing was done with a 384-pinhead printer, consisting of regular 3 × 3 spots per pin, yielding a total of 3456 available positions. Gene probes were printed in a single replicate. 2390 positions were used for probes, the remaining positions being empty. Membranes were kept humid by setting them onto three Hybond blotting-paper sheets soaked in denaturing solution (1.5M NaCl, 0.5M NaOH). Solid pins of 0.4-mm diameter were used. These pins spot 20 nl each time. PCR products were spotted five times so that each spot contained 20–30 ng DNA in a diameter of approximately 0.6 mm. After printing, the membrane was neutralized with 1.5 M NaCl, 0.5 M Tris/HCl (pH 7.2), 1 mM EDTA (pH 8.0) for 1 min and membranes were kept on filter paper until complete dryness.
Description
Nylon membrane (11 x 9 cm) with spotted PCR products up to 2.5 Kb, representing the full-length genes or a 2.5 kb internal fragment for those genes exceeding 2.5 kb for 2390 L.casei genes.
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