Construction of YAC libraries with large inserts.
The yeast artificial chromosome (YAC) cloning system makes it possible to clone large pieces of genomic DNA into yeast. Libraries have been made containing clones with inserts in the megabase-pair range. The basic protocol in this unit describes preparation of YAC vectors and transformation of ligated DNA into yeast spheroplasts. A support protocol describes titration of Lyticase to make spheroplasts. Additional support protocols detail two methods for partial digestion of genomic DNA: EcoRI restriction endonuclease-EcoRI methylase competition and the partial digestion of genomic DNA by use of limiting amounts of Mg2+, respectively.
Foote
Simon
S
Whitehead Institute, Cambridge, Massachusetts, USA.
Denny
Christopher
C
D000818:Animals; D018244:Chromosomes, Artificial, Yeast / Q000235:genetics*; D003001:Cloning, Molecular; D004274:DNA, Recombinant / Q000235:genetics / Q000302:isolation & purification; D004926:Escherichia coli / Q000235:genetics; D005821:Genetic Techniques; D005822:Genetic Vectors; D005826:Genetics, Medical; D015698:Genomic Library; D006801:Humans; D013104:Spheroplasts / Q000235:genetics; D014170:Transformation, Genetic
D004274:DNA, Recombinant