PFGE and YACs

Department of Research shipman at ubaclu.unibas.ch
Fri Oct 14 11:37:13 EST 1994


Hi Beth and "YAC" friend

Saw your query on the bionet and thought I would venture my "2 cents worth".
I've been isolating YACs from the ICRF 4X and 4Y genomic DNA libraries to
use as templates in cDNA direct selection protocols so I've already done a
few of the steps your friend would like to do/try. The YACs I've got are
900, 700, 400 and 250kb and the following procedures work well with all of
them. I'm not sure what your particular application with the purified YAC
will be, so i'll have to rely on your response if you require more
definitive protocols.
Extracting the YAC from 1% MP agarose (Boehringer Mannheim) was performed
after ethidium bromide staining and cutting out the YAC band using either
GeneClean (bio101) or Qaiex (Qaigen) according to the manufacturers
instructions. Both products have a suggested maximum capacity per 10ul of
beads, I simply used twice as much as recommended for the
extraction/purification. To my surprise the YACs remained intact after
both procedures..granted i only analysed an aliquot of the total isolate
but the predominant band of the expected size was present after PFGE. I
usually prepare my YAC from a 1cm "patch colony" on YPD plates using a
"rapid YAC DNA plug prep" and run the entire prep on PFGE. The yield from
such preps of YAC DNA is usually 100-300ng/ul.
Southern blotting to detect the YAC is always a good idea, if not an
obligatory control. Depends on how easy it is to separate your YAC from
the other yeast chromosomes. A southern would verify the presence of the
YAC and also indicate whether the YAC co-migrates with a non-YAC yeast
chromosome. Should you require more detailed protocols, don't hesitate to
get in touch...bon chance

Rob Shipman
Molecular Oncology
Lab 405 (ZLF)
University Hospital Research Center       tel 061 265 23 87
Basel, Switzerland                        fax 061 265 23 50
                                          email shipman at ubaclu.unibas.ch
 
In article <379ucc$q3q at quartz.ucs.ualberta.ca>,
erosenbe at gpu.srv.ualberta.ca (Elizabeth Rosenberg) wrote:

> Hi all!
> 
> I am posting this for a friend, and I don't work with yeast at all myself 
> although we do use PFGE extensively.  She would like to use PFGE to 
> identify, characterize and isolate YAC chromosomes.  Although I can help 
> her with the various considerations for PFGE, she would like some 
> information about the possibility of extracting the YAC from the gel 
> itself.  Does anyone have any protocols for this?  Also, what sort of 
> quantities would you be able to recover this way?  In addition, how 
> practical would it be to use Southern blotting on these gels specifically 
> for the YAC?  Would the signal be large enough to detect?  I know these 
> are general questions, but any suggestions are appreciated.  I'm 
> currently having intermittent problems with my e-mail, so if you could 
> post responses to the group for now so that I can read them.
> 
> Thanks,  Beth
> 
> --
> _____________________
> Beth Rosenberg                    erosenbe at gpu.srv.ualberta.ca
> Cross Cancer Institute
> University of Alberta



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