( reviously described). The other half wa...)Human Universal cDNA Library Array I

reviously described). The other half was used for a second round of subtractive hybridization resulting in a library of 2.5 x 10⁴ clones (Figure 2). These two normalized libraries were directly amplified on agar plates. The individualized colonies were lifted onto membranes and duplicated onto a second set of membranes. One set was used to purify the DNA for analysis and storage while the other set was soaked in a sucrose solution and frozen at -80C to be used for further organization into microwell plates.

Figure 2

Organization in Microwell Plates and DNA Preparation

Figure 3

The frozen membranes were thawed on agar plates, and the colonies individually picked and cultured in microwell plates to be finally stored at -80C. Approximately 123,000 and 22,000 clones were arrayed from the two libraries obtained after one and two cycles of normalization, respectively. The DNA analysis of individual clones picked at random revealed an average insert length of 1.7 kb (Figure 3), which was consistent with an earlier analysis performed on over 100 clones (data not shown).

To preserve the advantage of having organized primary clones that had never been in contact with each other, the subsequent amplifications of these clones were performed by growing the colonies separately before any pooling process. The bacterial colonies were individually grown on agar plates with 96 clones per plate. The clones from each plate were then pooled to isolate the DNA, and the DNA samples obtained from the resulting 1,536 pools were double-spotted onto nylon membranes for the screen
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