Fast Decrosslinking - (May/02/2010 )

Hello,
I was wondering if anyone had a relatively fast way to decrosslink following sonication. I like to check the size of my chromatin before I proceed to the IP, but I currently use proteinase K O/N. I have seen some mention of Chelex 100 on these forums, but if anyone has a quick method they can post that would be great. --- I am only concerned with seeing how well my chromatin is sonicated. I would continue to do RNAase A tx, and proteinase k O/N after the IP since that works well for me.

Thanks

Chipper on May 2 2010, 03:15 PM said:

Basically add your chromatin to 100µl 10% chelex suspension and add 20µg proteinase K. Digest at 50-60C for 15 minutes and then heat to 95-100C for 10-15 minutes. Then just run the supernatant on a gel. You can use this method for preparing ChIP samples as well (just add the chelex and proteinase K to the beads after the last wash of the IP) but keep in mind the end product is mostly single stranded DNA and thus not a good template for adding adapters to do ChIP-chip or ChIP-seq. Works great for PCR though. Oh, and if you're trying to be quantitative, you might try rinsing the beads/slurry with nuclease free H2O after removing the supernatant and combining the rinse with the supernatant.

Thanks, I'll give it a go!

Chipper on May 2 2010, 06:15 PM said:

I typically do the following to check for good sonication:

Remove 5 uL of sample
Bring to 50 uL volume in elution buffer
Add proteinase K and incubate for 2hrs at 62C
Purify using spin columns
Run on gel

I don't think proteinase K is necessary to do O/N, especially when just checking sonication conditions.

MM