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sonication in ChIP / nonReverse


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#1 BioBus

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Posted 28 June 2009 - 06:07 AM

Hi ChIPpest,

Could I use the non- reverse sonicated aliquot on Agarose gel to check the sonication efficiency ?

If remeber correctly , at least one plant ChIP in Nature protocols just check the sonication
efficiency with non- reverse Agarose gel ( an aliquot , 5-10 % of quantity of IP chromatin )

Like gel shift , it is understandable that non reversed DNA could appear up- shift
in comparison with the reversing.

The picture attached is non- reversed , could it work or further sonication needed?

The bottom smear ( < 300 bp) could be RNAs, but the the > 500 bp could be the
fragmented DNAs or just something messy ?? ( in this forum, on post reply tells that
SDS can bind EtBr. an dhere 1% SDS in the lysis buffer used ).



If it could work ; it makes thing simpler and save time.

Any suggestion is greatly appreciated.

bioBus

Attached Thumbnails

  • nonrev1.png

Edited by BioBus, 28 June 2009 - 06:19 AM.


#2 TracyDuke

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Posted 29 June 2009 - 10:23 AM

You can do a quick Proteinase K digestion at 45 degree for 1 hour. Then you run it on the gel, it appears almost as good as after revers cross-linK plus purification.




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