Laboratory for Functional Genome Analysis (LAFUGA) - Genomics Unit, Gene Center of the LMU Munich, Munich, Germany
Manufacture protocol
Fifteen microliters of PCR reactions of amplified cDNA clones were transferred to 384 well microtiter plates (AbGene, Epsom, Surrey, UK) containing 15 µl of 2 x spotting buffer (40 mmol/l Tris-HCl pH 8, 2 M NaCl, 2 mmol/l EDTA, and bromphenol blue). PCR products were spotted onto amphoterous nylon membranes (Nytran plus, Schleicher & Schuell, Dassel, Germany) within an area of 2 x 5 cm. For spotting an Omnigrid Accent microarrayer (GeneMachines, San Carlos, CA) was used with solid pins (0.015 inches in diameter, SSP015; Telechem International; Sunnyvale, CA). Spotting was performed 6 times for each PCR product on the same position for sufficient and equal application. Twenty-eight arrays were produced simultaneously. The spotted DNA was denatured with 0.5 N NaOH for 20 min at room temperature. DNA was immobilized by baking for 30 min at 80 °C and UV crosslinking (120 mJ/cm2).
Messenger RNA profiling of bovine endometrium during the estrous cycle using a custom-made cDNA array
Data table header descriptions
ID
cDNA /oligo ID
Internal cDNA clone ID or Operon oligo ID
POSITION
Position on the array (subarray row_subarray column_row_column)
ORF
Entrez Gene ID of the putative human ortholog
GENE SYMBOL
Official Gene Symbol of the bovine gene or of the human ortholog or both if different
TOP_BLAST_HITS
GenBank accession number of best Blast hit
COMMENT
df: cDNA clone consists of two artificially ligated cDNA fragments of two different cDNAs (derived from different mRNAs); np: PCR reaction failed, no specific product visible on agarose gel
GB_ACC
Accession number of cDNA sequence in GenBank est division
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