ZFNs can be used to generate targeted genomic deletions of large segments of DNA in a wide variety of cell types and organisms.
In the August issue of the journal Cold Spring Harbor Protocols (http://cshprotocols.cshlp.org/TOCs/toc8_10.dtl), Jin-Soo Kim and colleagues (http://chem.snu.ac.kr/eng/Faculty/faculty_detail.asp?seqno=1015&link=faculty) present "Analysis of Targeted Chromosomal Deletions Induced by Zinc Finger Nucleases," a detailed protocol for the detection and analysis of large genomic deletions in cultured cells introduced by the expression of ZFNs. The method described allows researchers to detect and estimate the frequency of ZFN-induced genomic deletions by simple PCR-based methods. This featured protocol is freely available on the journal's website (http://cshprotocols.cshlp.org/cgi/content/full/2010/8/pdb.prot5477).
Immunoprecipitation is a commonly used technique for isolating and purifying a protein of interest. An antibody for the protein is incubated with a cell extract, and the resulting antibody/antigen complex is pulled out of solution. The method used for preparation of the cell extract can be critical for the experiment's success. The choice of lysis conditions must be tailored to the nature of the epitope recognized by the immunoprecipitating antibody. "Lysis of Cultured Cells for Immunoprecipitation," featured in the August issue of Cold Spring Harbor Protocols (http://cshprotocols.cshlp.org/TOCs/toc8_10.dtl), provides detailed instructions for the lysis of cells grown as monolayer cultures and cells grown in suspension. The protocol offers a detailed comparison between different commonly used lysis buffers and protease inhibitor cocktails, as well as a guide to preparing a general protease inhibitor cocktail. The article is freely available on the journal's website (http://cshprotocols.cshlp.org/cgi/content/full/2010/8/pdb.prot5466).
About Cold Spring Harbor Protocols: Cold Spring Harbor Protocols (www.cshprotocols.org) is a monthly peer-reviewed journal of methods used in a wide range of biology laboratories. It is structured to be highly interactive, with each protocol cross-linked to related methods, descriptive information panels, and illustrative material to maximize the total information available to investigators. Each protocol is clearly presented and designed for easy use at the bench—complete with reagents, equipment, and recipe lists. Life science researchers can access the entire collection via institutional site licenses, and can add their suggestions and comments to further refine the techniques.
About Cold Spring Harbor Laboratory Press: Cold Spring Harbor Laboratory Press is an internationally renowned publisher of books, journals, and electronic media, located on Long Island, New York. Since 1933, it has furthered the advance and spread of scientific knowledge in all areas of genetics and molecular biology, including cancer biology, plant science, bioinformatics, and neurobiology. It is a division of Cold Spring Harbor Laboratory, an innovator in life science research and the education of scientists, students, and the public. For more information, visit www.cshlpress.com.
David Crotty | EurekAlert!
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Enzymes behave differently in a test tube compared with the molecular scrum of a living cell. Chemists from the University of Basel have now been able to...
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