ChIP has eaten all my DNA! - (Nov/01/2007 )
I used Upstate EZ ChIP kit (17-731) to do chip on two human cell lines, after a first trail experiment no DNA was persent in final product, and no PCR product either. So I checked step by step. First is sonication, I used a misonix microson XL2000-010 sonicator with a 1/8 inch tip, I used 1ml SDS lysis buffer for 10e7 cells and sonicated in 350ul volume. The power setting is 5 (7wt) and I sonicated for 7 times(10 sec each with 5 sec interval) on ice. I reversed the crosslink and ran 20ul of chromatin on gel. The smear has a right range of 200-1000bp, but two strange bands appear in all samples (including unsheared control). And PCR use provided postive control did not bring a thing. (Only have a band in genomic DNA postive control) Where did all DNA gone?? Did I sonicat too much?
Thanks all for your attention.
for the gel picture
first lane is 100bp ladder
2~7 sonicated 4 to seven times (crosslinked)
8 unsheared control
9-13 unlinked chromatin sheared 4-7 times
14 DL 2000 DNA marker (100 250 500 750 1000 2000bp)