Smear in PCR after ChIP - (Aug/23/2007 )
Please help!
I've been doing ChIP for months now and was getting nice results, then all of a sudden I started getting smears after my PCR. I know there's nothing wrong with the primers or the master mix as I include a positive control from a ChIP that worked which comes out as a bright specific band. Here's a brief outline of my ChIP (which basically follows the Upstate protocol):
Crosslink at 37C for 10 minutes with 1% FA. Quench with 0.125M Glycine.
Wash 2x ice cold PBS
Resuspend in 400ul SDS lysis buffer and sonicate a 10um with a Sanyo Soniprep 150 5x on ice, 10 secs on 10 secs off.
Pre-clear 1 hour with 80ul pre-blocked protein G agarose with ssDNA (100ug/ml) and 1% BSA.
Add 2ug ab for 4hrs at 4C.
Add 60ul beads to precipitate.
Wash beads 1x Low salt, 2x high salt, 1x LiCl, 2xTE.
Elute with 1% SDS, 1M NaHCO3.
Reverse crosslinks with NaCl 4hrs - overnight.
Precipitate DNA with phenol/chloroform.
Quantitate DNA using nanodrop and add 40ng to PCR.
The strange thing that I've also noticed is that when I run 5ug of my sonicated DNA on a gel, I get some high molecular weight unsheared DNA and also a smear at around 500bp. However, I've also run my DNA using the Agilent Bioanalyzer to check my fragment lengths and this shows DNA at 1500bp! I'm confused! Please help anyone!
Many thanks,
Are you sure it is smear? I guess that is probably your chromatin DNA. I had same problem as I just posted. Actually, mine doesn't like smear, after I load my samples without PCR(equal amount of DNA), I got the same smear as PCR one.
I've been doing ChIP for months now and was getting nice results, then all of a sudden I started getting smears after my PCR. I know there's nothing wrong with the primers or the master mix as I include a positive control from a ChIP that worked which comes out as a bright specific band. Here's a brief outline of my ChIP (which basically follows the Upstate protocol):
Crosslink at 37C for 10 minutes with 1% FA. Quench with 0.125M Glycine.
Wash 2x ice cold PBS
Resuspend in 400ul SDS lysis buffer and sonicate a 10um with a Sanyo Soniprep 150 5x on ice, 10 secs on 10 secs off.
Pre-clear 1 hour with 80ul pre-blocked protein G agarose with ssDNA (100ug/ml) and 1% BSA.
Add 2ug ab for 4hrs at 4C.
Add 60ul beads to precipitate.
Wash beads 1x Low salt, 2x high salt, 1x LiCl, 2xTE.
Elute with 1% SDS, 1M NaHCO3.
Reverse crosslinks with NaCl 4hrs - overnight.
Precipitate DNA with phenol/chloroform.
Quantitate DNA using nanodrop and add 40ng to PCR.
The strange thing that I've also noticed is that when I run 5ug of my sonicated DNA on a gel, I get some high molecular weight unsheared DNA and also a smear at around 500bp. However, I've also run my DNA using the Agilent Bioanalyzer to check my fragment lengths and this shows DNA at 1500bp! I'm confused! Please help anyone!
Many thanks,
Is it possible to pull down that much chromatin DNA after IP so that you see it on the gel (after PCR)?
Thanx