membrane baking-why bother?

daj (David Johnston) daj at nhm.ic.ac.uk
Thu Feb 17 03:23:41 EST 1994


On 16 Feb 94 13:53:31 EST,
  <lappel at eagle.wesleyan.edu> writes:

>In article <CL8Jqw.HBn at ucc.su.OZ.AU>, Mark Smith writes:
>> In article <2jeh4l$bcq at nic.umass.edu> AMRUT at UCSVAX.UCS.UMASS.EDU (AMRUT S BHOGLE) writes:
>>> 
>>>A friend of mine wants me to find out if nylon membranes can be baked without
>>>a vacuum to cross-link DNA onto the membranes.  
>>>
>>>Thanks!!
>> Yes they sure can. That is unless they are nylon reinforce nitrocellulose.
>> We bake for 2-18hr at 80C.
>> Mark Smith
>> Dept. Biochemistry
>> Uni of Sydney
>> Sydney, 2006
>> mts at biochem.su.oz.au
> We baked nitrocellulose and nylon membranes for years without vaccuum
>(not having a vaccuum oven, we just tried without) with no trouble.  The only
>time anyone had trouble was when one guy caught a corner of his dry blot under
>the heat sealer element when sealing it into seal-a-meal, and ended up with a
>bag of smoke. (nitrocellulose is the same thing they sell in magic/hobby
>supply stores as flash paper, also known as gun-cotton or something like that)
> However, these days, most people use UV crosslinking instead of baking:
>Stratalinker or the like.  Much faster than the old 2 hours or until-you
>remember-to-take-it-out bake.
> For library screening, the fastest method is to do your lambda-lifts
>onto a nylon membrane of some sort (plain nitrocellulose is rumored Not to work
>this way) put the filters straight into a Whatman or cheapo-filter paper
>sandwich, and autoclave at 212C for 2 minutes.  Opens the phage and sticks it
>to the filter without base-neutral-SSC rinses or having to bake. Much faster
>for chromosome walking.
> 
> Laurel F. Appel    (LAPPEL at EAGLE.WESLEYAN.EDU)
> Genetics Group
> Biology Dept.
> Wesleyan University
> Middletown CT 06459

No vacuum oven? Chances are though that you have a heated gel drier. 
Use that instead if you want to bake NC. Works a treat.

(Details: lay NC on clean, smooth filter paper (eg Whatman no 1), cover 
with clingfilm and away you go. You may find that the filters adhere to the 
filter paper but can easily be released by moistening the filter paper with 
distilled H2O or SSC etc.)
Cheers,
DAJ

David A. Johnston
Dept of Zoology, The Natural History Museum, Cromwell Road,
South Kensington, London SW7 5DB.
(tel 071 9389297, fax 071 9388754, email daj at nhm.ic.ac.uk)



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