UNIT 5.7 Purification and Characterization of YACs Containing Large Inserts
Published Online: 1 MAY 2001
Copyright © 2003 by John Wiley and Sons, Inc.
Lab Protocol Title
Current Protocols in Human Genetics
How to Cite
Gemmill, R. M., Bolin, R., Strauss, W. M. and Pavan, W. 2001. Purification and Characterization of YACs Containing Large Inserts. Current Protocols in Human Genetics. 00:5.7:5.7.1–5.7.20.
- Published Online: 1 MAY 2001
- Published Print: FEB 1994
This unit provides protocols for characterizing DNA segments cloned in YACs and for purifying YACs from yeast chromosomes. The first basic protocol describes Southern blotting and partial-digest restriction analysis of YACs. These methods are useful for determining the size and complexity of the cloned insert DNA, the presence and location of particular restriction sites or sequences, and even the species of origin of the insert DNA (indicated by hybridization to species-specific repetitive elements such as Alu repeats). The second basic protocol describes gel purification of YACs for use in procedures requiring pure YAC DNA, such as mammalian-cell transformation and subcloning into smaller insert vectors. The third basic protocol details characterizing and analyzing YACs: in vivo fragmentation via homologous recombination with specialized fragmentation vectors containing specific probe sequences or repetitive elements, followed by Southern blotting with YAC- and human-derived probes.