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Human Universal cDNA Library Array I

ing procedure (Figure 4).

Figure 4

Screening for Human b-actin Clones

The presence of the ubiquitous b-actin gene (1.8 kb)4 was analyzed in each library to evaluate the efficiency of the fractionation-normalization procedures. In this analysis, we probed the libraries with two different radiolabeled oligonucleotides.5 One probe was designed to hybridize to the beginning of the 5 translated region (ATG included) while the other corresponded to the end of the 3 untranslated region (Figure 5 and Figure 6). The percentage of full-length cDNAs was determined by comparing the two results.

Figure 5

In situ hybridization of 6,000 colonies from the library obtained before fractionation established that b-actin was originally represented at approximately 1 in 200 clones with 45% being full-length (data not shown). Dot blot analysis on the Master Membranes (Figure 6) showed that after fractionation and one round of normalization, the representation of this gene fell to 1 in 5,000 clones with 70% being full length. After the second round of normalization no b-actin clone was detected in the organized colonies, suggesting that, after extensive hybridization, each of the 22,000 clones arrayed could be unique.

Stratagene Now Provides These Membranes for Screening Procedures

Figure 6

A set of four Master Membranes, each the size of a microwell plate, allows the screening of 1,536 pools of 96 clones in the same conditions as ordinarily
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