Efficient procedure for preparing cosmid libraries from microgram quantities of genomic DNA fragments size fractionated by gel electrophoresis.
Publication/Presentation Date
10-1-1990
Abstract
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.
Volume
10
Issue
5
First Page
279
Last Page
284
ISSN
0934-8832
Published In/Presented At
Ala-Kokko, L., & Prockop, D. J. (1990). Efficient procedure for preparing cosmid libraries from microgram quantities of genomic DNA fragments size fractionated by gel electrophoresis. Matrix (Stuttgart, Germany), 10(5), 279–284. https://doi.org/10.1016/s0934-8832(11)80182-4
Disciplines
Medicine and Health Sciences
PubMedID
2084510
Department(s)
Department of Pathology and Laboratory Medicine
Document Type
Article