BIIIIG problem with top-agarose

Bill Alexander ALEXANDERW at cber.cber.fda.gov
Mon Nov 6 12:10:45 EST 1995


>
>In article <47fj07$n4v at due.unit.no>,
>Hilde Nilsen  <hilden at unigen.unit.no> wrote:
>>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.
>
>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.
>
>- -pjf
>
Maniatis also used to suggest using Agarose instead of agar to make your top
agar.
Regards,
 
Bill Alexander                                
ALEXANDERW at cber.cber.fda.gov                                                    

     "640K ought to be enough for anybody." -- Bill Gates, 1981



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