BIIIIG problem with top-agarose

Harry Witchel Harry.Witchel at bristol.ac.uk
Mon Nov 6 11:00:09 EST 1995


In article <47fj07$n4v at due.unit.no>, hilden at unigen.unit.no says...
>
>Hallo reader
>
>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.
>
>Please  help me!
>
>My e-mail address: hilde.nilsen at unigen.unit.no
>
>
>
As pointed out by Zinc, for library transfer to be successful:

1)  bottom agar must be very dry before pouring the top agarose
2)  the top agarose must be cold and hard before application of filter.

Another way of drying out the bottom agar is to warm the plates for 1-3 
hours in a 37C incubator upside down (ie agar near ceiling) with the main 
plate halfway off the lid (so moisture can escape).




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