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Proteinase K after Sonication - (Jun/10/2009 )

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As we up going to increase our ChIP numbers soon I thought I would try your fast reversal method. Who do you buy your Chelex from?

You should proteinase K treat and do the reversal of crosslinking before running on the gel. You don't have to RNase treat but if you don't then you'll see a fairly bright RNA smear in the 100-200bp region on your gel.

If you want to do a fast reversal of crosslinking just dilute your chromatin (10-20ul) in 100ul of 10% chelex-100 suspension. Add proteinase K and digest for 15 minutes at 50-55C and then boil or heat on a heat block at 100C for 10minutes. Then run the supernatant on the gel. This works for ChIP too. Just add the 100ul chelex suspension to your protein A beads after the last wash and proceed as above. Works great for either agarose or magnetic beads.


KPDE on Jul 16 2009, 04:09 PM said:

zyka on Jul 16 2009, 12:49 PM said:

Hi guys,

My question might sound silly but... Why do you put proteinase K in your samples at the sonication step? Is it because you're working with cells? We are sonicating genomic DNA extracted from blood, do we need to add proteinase K as well??


If you're planning on using the chromatin for ChIP you don't use the proteinase K until after you've run the IP. You definitely want as little proteolysis as possible before and during the IP.

Ok. Our IP protocol includes proteinase K after binding of the antibody with the 5methyl-cytosines.

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