By Peter G. Stockley (auth.), Benoît Leblanc, Tom Moss (eds.)
Gene expression can suggest the variation among a practical and non-functional genome, among health and wellbeing and disorder, and with the improvement of transgenic plants, the variation among survival and hunger. In DNA-Protein Interactions: ideas and Protocols, 3rd Edition, this important topic is stated to this point with protocols exploring the main state of the art advancements within the box, together with in vivo and genome-wide interplay options. Addressing issues corresponding to chromatin immunoprecipitation, topological reports, photocrosslinking, agonize and imaging recommendations, the quantity totally updates and expands upon the winning past variants. Written within the handy and informative Methods in Molecular Biology™ sequence structure, chapters contain introductions to their respective matters, lists of the required fabrics and reagents, step by step, comfortably reproducible laboratory protocols, and notes on troubleshooting and heading off identified pitfalls.
Comprehensive and authoritative, DNA-Protein Interactions: rules and Protocols, 3rd Edition serves as an incredible consultant for all these exploring this dynamic, crucial, and more and more cheap zone of research.
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Extra info for DNA-Protein Interactions: Principles and Protocols, Third Edition
And Maniatis, T. (1989). Molecular Cloning, A Laboratory Manual. Cold Spring Harbor, New York, NY. 50. , Brisson, I. L. (1993). A simple apparatus for fast and inexpensive recovery of DNA from polyacrylamide gels. Biotechniques 14, 942–948. 51. , Giasson, M. L. (1996). Multiple nuclear regulatory proteins bind a single cis-acting promoter element to control basal transcription of the human alpha 4 integrin gene in corneal epithelial cells. DNA Cell Biol. 15, 779–792. 52. , Eskild, W. L. (1997).
A careful titration of the DNase concentration is therefore essential to optimize the detection of a footprint and can even make the difference between the detection of a given interaction or lack thereof. The following protocol was developed to study the footprinting of the Xenopus ribosomal transcription factor xUBF, which is a rather weak DNA-binding protein with a rather broad specificity.
32 Gaudreault et al. 14. Although 5¢ end-labeling of the selected DNA fragment is best done using polynucleotide kinase, very efficient labeling can also be accomplished using alternative procedures, such as filling 5¢-protruding ends using the Klenow fragment of E. coli DNA polymerase I (49), a particularly attractive alternative when crude nuclear extracts rich in various phosphatases are used (in the event that no phosphatase inhibitors are used in the binding buffer). Larger DNA segments can also be efficiently labeled by PCR.