Two-level polymerase chain reaction (PCR) amplifications were used to prepare DNA for spotting. BAC DNAs were extracted using Millipore (Billerica, MA) Montage BAC96 miniprep kit following supplier instructions after culture in 96-well plates with 1.5 mL 2× LB medium. Each BAC was then amplified by two independent degenerate oligonucleotide-primed polymerase chain reaction (DOP-PCR) procedures using two different primers: 6MW and DOP2. About 75 ng of DNA of the primary DOP-PCR products was then amplified through a secondary PCR. Finally, PCR products corresponding to a single BAC were pooled, ethanol-precipitated, dried, and resuspended in 75% formamide and 25% water for spotting using a Microgrid II automated microarrayer (Biorobotics; Genomic Solutions, Ann Arbor, MI) on GAPS II slides (Corning Life Sciences, Acton, MA).
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