Subcloning of YACs

Ola Myklebost olam at radium.uio.no
Fri Apr 16 06:54:38 EST 1993


Somebody asked about subcloning of YACs, and my reply yesterday got lost
due to a local mail server problem.

The strategy used was YAC purification by PFGE in LMT agarose.

I went to the Cold Spring Harbor course on YAC manipulation last fall
(which I highly recommend), and we were then advised to rather subclone the
entire yeast + YAC DNA in lambda (or cosmids). The yield from PFGE
purification is low and the quality poor. Thus you cannot very well control
the conditions for library construction.

However, a total library is easy to make (at least in lambda), and it
doesn't need to be large to cover a yeast+YAC genome. Clones are then
screened with total human (or other) DNA as probe (or pBR fragments to get
YAC ends).
 
Ola Myklebost             Email olam at radium.uio.no
Dept of Tumor Biology
Inst for Cancer Research
The Norwegian Radium Hospital
N-0310 OSLO, Norway
Tel +47-22-50-60-50, xtn 9830, Fax +47-22-52-24-21



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