Extraction of products from in situ PCR

David Titus davidt at mjr.com
Tue Jan 28 17:03:58 EST 1997


> Can anyone help with a method for extraction of PCR 
> products from in situ PCR slides for subsequent agarose 
> gel electrophoresis. 


It is often desirable to recover some of the liquid reaction from a
microscope slide after an in situ thermal cycling protocol has been
completed.  Here are two ways to accomplish this.

1. Using an adhesive-backed chamber.
One approach is to use a chamber affixed to the slide and designed to
withstand the temperature extremes of the protocol.  For example, if you
are doing  thermal cycling  with denaturing temperatures over about
800C,  the chamber must use a special adhesive which won't fail in the
elevated pressure and hot water environment within the chamber. 

These chambers are generally two-piece units consisting of a "frame"
adhered to the slide and a thin plastic cover piece put in place after
the reagent is added to the chamber.* When the thermal cycling protocol
is complete,  simply cut the plastic cover piece open with a razor blade
and recover the contents with a pipet. 

2. Using a glass cover slip.
An alternative approach is to recover reaction liquid from under a
standard cover glass.  This takes a bit of practice but isn't too
difficult. 

If the cover has been sealed with nail polish, the seal must first be
softened by soaking in ethanol or xylene.  The softened "bead" of polish
is then carefully removed with a razor blade, leaving the cover in
place.

If the cover has been sealed with Self-Seal Reagent**,  soften the seal
by placing the slide between two sheets of damp filter paper for a
minute or two.  

After softening the seal layer, or removing the nail polish,  remove the
cover as follows.   Hold the slide (with cover glass in place) with one
hand at the frosted end and rest the other end on a fresh piece of
Parafilm on the bench top, cover slip surface facing you.  Using a fresh
razor blade or scalpel, carefully lift the cover off,  beginning at an
upper corner.    As the cover is loosened, open it away from the slide
to approximately 45 degrees as if it were hinged along one of the long
edges. Much of the internal liquid will accumulate by the Parafilm in
the crease between the slide and the cover and can be recovered using a
pipet.  As the cover comes off you can use your thumb to hold it away
from the slide while progressively loosening it with the razor blade.
(Wear eye protection!) 

*One frame-type device for sealing in situ reactions on slides is the
Frame-Seal Chamber from MJ Research.
**Self-Seal Reagent (also from MJ Research) is a reagent that is added
directly to the reaction mix and forms a seal around the periphery of
the cover glass during elevated temperature incubations. If using
Self-Seal Reagent, it is recommended that the recovered material be
diluted with an equal volume of gel loading buffer prior to loading on a
gel.

David Titus
davidt at mjr.com




More information about the Methods mailing list