BIIIIG problem with top-agarose

[bio.tamu.edu]

> >I am screening a genomic lambda libary. I am using host cells without
selection.
> >The problem is that as I am transferring the plaques to Hybond N+ membranes, 
> >the top-agarose adheres to the membrane thereby ruining everything.
> >I suspect this to happen because of moisture between the top-agarose
and the medium.

> >So, what can I do to prevent this? I am cooling the medium to 50 deg.
celsius before
> >preparing the plates so too hot medium should not be the problem.

> i have two suggestions for you.

> 1) try to use plates that have been 'cured' for a day or two so that
> they are not to wet when you apply the top agar.

> 2) this is what i usually do to prevent the top agar from lifting off.
> i just cool the plates to 4 deg C after the plaques have grown and i
> have not had any problems with it lifting off when i do this.


I would just add that it is best to cool at 4 degrees for a minimum of 2
hours to be safe, before doing the lifts.

Kim Snowden
ksnowden at bio.tamu.edu