Purifying YAC DNA
ANDREI.POPOV at bbsrc.ac.uk
Sat Nov 4 13:01:00 EST 1995
>We are hoping to extract high enough quality YAC DNA (pure) from PFGE gels for
>subsequent restriction digestion. Contemporary proeedure (apparently) is to
>make a total library of yeast and YAC DNA, screen with COT-1, and pick these
>clones. This seems a considerable amount of work.
>So are there any decent protocols that get around this. How about Agarase (or
>is that only for LMP agarose).
though it is not clear what exactly you intend to do with this
DNA I can suggest the following:
cut the YAC band from the agarose gel (you can stain with EtBr
the outer lanes and cut afterwards the central band corresponding to your YAC)
, equilibrate in the restriction buffer, restrict and go on.
beta-Agarase indeed works better if the gel slice was melted before
the addition of the enzyme.
For construction of sublibararies you do not even need to get rid
of the LMA; all the manipulations can be done in agarose and
it is even very convenient for buffer changes, just cool the agarose
and dialyse for a while.
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