Efficient procedure for preparing cosmid libraries from microgram quantities of genomic DNA fragments size fractionated by gel electrophoresis.
A modified procedure for preparing cosmid libraries from genomic DNA is described. Genomic DNA was partially digested with a restriction endonuclease, and DNA fragments of appropriate size fractionated by agarose gel electrophoresis. A cosmid library was prepared, prescreened, and used to isolate gene inserts with previously published procedures. In one series of experiments, a modified cosmid vector containing stuffer fragments was used to prepare cosmid libraries containing partial SphI digests of 25 to 35 kb. From 10(5) to 10(7) clones were obtained per microgram of size-fractionated genomic DNA. From 10 to 100 hybridization-positive clones of a single copy gene (COL2A1) were obtained from plates that were positive in the prescreening step. Restriction mapping of over 20 clones and nucleotide sequencing of over 20,000 bp in each of two clones indicated that the inserts were faithful copies of the gene. In another experiment, a standard cosmid vector was used to prepare a cosmid library containing partial BamHI fragments of 30 to 45 kb. Genomic libraries can be prepared with 5 to 20 micrograms of genomic DNA and a large number of clones containing 25 to 45 kb fragments of a single copy gene can be isolated in about three weeks.
Ala-Kokko
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Department of Biochemistry and Molecular Biology, Jefferson Institute of Molecular Medicine, Jefferson Medical College, Thomas Jefferson University, Philadelphia, PA 19107.
Prockop
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DJ
D003001:Cloning, Molecular; D003094:Collagen / Q000235:genetics; D003360:Cosmids; D004247:DNA / Q000235:genetics / Q000302:isolation & purification*; D015252:Deoxyribonucleases, Type II Site-Specific; D004587:Electrophoresis, Agar Gel; D015723:Gene Library; D005796:Genes; D006801:Humans
D003094:Collagen; D004247:DNA; C046184:endodeoxyribonuclease PaeI; D015252:Deoxyribonucleases, Type II Site-Specific