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

Hi KPDE,

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?
Thanks!
Clare


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.