Please check my ChIP sonication gel - (Dec/20/2006 )
Would anyone tell me why I'd have a higher molecular weight group and a lower molecular weight group after sonication? I use 400ul cell lysate to do sonication.
And the lysis buffer composition is as following:
50 mM Hepes pH 7.9
140 mM NaCl
1 mM EDTA
1% Triton X-100
Protease inhibitor coctail (Roche)
I use fisher scientific sonic dismembrator. I have to say the IP of polymerase II antibody and PCR with primers amplifying GAPDH promoter region works terrific, I got a Ct value around 24 and nothing in mouse IgG control after 32 cycles. But I'm concerned about the higher weight DNA. How I can get rid of them?
Thank you for any input!
Are you talking about the smear? If so, we already discussed about this in the forum... It could be RNA if you didn't RNase-treated your samples before loading onto the gel
oh, thank you. I did treat cells with Rnase...... And what I'm worrying about is those that are bigger than 1000bp.
Do yo have a lot of insoluble material after sonication (i.e. is the lysate cloudy after sonication). I had this problem before I standardized the number of cells I used in a sonication. When I had larger nuclear pellets the lysate was cloudy after sonication and I had the smear at high and low molecular weight (and as in your case, the low MW smear was not RNA). When I had a smaller nuclear pellet the lysate was clear after sonication (to my eye anyway) and the high MW smear was not present. So I found that if I just increased the sonication time till the lysate was clear, regardless of the size of the nuclear pellet, then the high MW smear disappeared.
Thank you, KPDE. I'll try!