Processing of cDNA and genomic kilobase-size clones for massive screening, mapping and sequencing by hybridization.
Efficient procedures for managing a large number of M13 or plasmid clones have been developed. In addition to picking, clones are directly arrayed in multiwell plates by dispensing diluted transformation mixtures. Metal pin arrays are used for fast inoculations of preparative plates filled by medium or by PCR mixture. Growth of M13 clones in multiwell plates is optimized to obtain a consistently high yield, and a PCR protocol is defined for reliable amplification of several thousand M13 or plasmid inserts per day in BioOvens. Over 80,000 cDNA inserts have been amplified. The phages or amplified inserts are spotted on nylon filters using an array of pins having a flat bottom, 0.3 mm in diameter. The procedures are suitable for an automated processing of hundreds of thousands of short clones from representative cDNA and genomic libraries. Hybridization of arrayed clones with oligonucleotide and complex probes can simplify the search for new genes and accelerate large-scale sequencing.
Drmanac
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Argonne National Laboratory, IL.
Drmanac
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D017104:Bacteriophage M13 / Q000235:genetics*; D001483:Base Sequence; D018076:DNA, Complementary / Q000737:chemistry / Q000235:genetics*; D008969:Molecular Sequence Data; D009693:Nucleic Acid Hybridization; D010957:Plasmids; D016133:Polymerase Chain Reaction
D018076:DNA, Complementary