UNIT 6.10 Analysis of Isolated YAC Clones
Published Online: 1 MAY 2001
Copyright © 2003 by John Wiley and Sons, Inc.
Lab Protocol Title
Current Protocols in Molecular Biology
How to Cite
Chaplin, D. D. and Brownstein, B. H. 2001. Analysis of Isolated YAC Clones. Current Protocols in Molecular Biology. 20:V:6.10:6.10.1–6.10.19.
- Published Online: 1 MAY 2001
- Published Print: OCT 1992
This unit provides a series of protocols describing the analysis and manipulation of an isolated YAC clone. The procedures are based upon the use of the YAC vector pYAC4. Once an isolated YAC clone has been obtained from a core laboratory, the clone can be analyzed as described herein. Methods for analysis involve growing and storing YAC-containing yeast strains and purifying YAC DNA in a form suitable for assessing the size of the artificial chromosome and for conventional Southern blotting. Preparation of yeast chromosomes in agarose plugs for subsequent analysis by pulsed-field gel electrophoresis is also described. Additional protocols are provided for recovering DNA fragments from the ends of a YAC genomic insert to be used as probes for detecting chimerism and for chromosome walking. Finally, preparation of high-molecular-weight YAC DNA is described and a general method for subcloning YAC inserts into cosmid or λ vectors for higher-resolution analysis is provided.