Strong bands at 220 bp after sonication - (Feb/15/2007 )
Hello,
I'm starting a ChIP experiment using mouse brain tissue. Briefly, fresh or frozen tissue is homogenized in cold 1xPBS, I cross link with 1% (total conc) formaldehyde for 10 and 20min at RT( some samples at 10 min, some for 20), quench crosslinking with 0.125M Gly, then incubate for 5min RT, then spin at 2k for 5 min to collect cells. I resuspend the cell pellet in 0.5ml SDS lysis buffer and incubate on ice for 10 min.
Then I sonicate using a Sonics vibracell130 (or something like that) with a 2mm probe, I sonicate at 30% amplitude. samples in a 1.5ml eppendorf are partially submersed in ice cold water during sonication. I've used several different sonicating procedures:
6 pulses for 25" with 60" rest between pulses
8 pulses, 10", 60" rest
10 pulses, 10", 60" rest
15 pulses, 10", 60" rest
No foaming occurs
After sonicating, I spin down the sonicates at 5k rpm, for 5 min, collect the supernatent then I aliquot the supe and apply some directly to a 1.5% agarose gel.
I know one should first add proteinase K, RNase, reverse crosslink, etc, but I was wondering if any of these steps are necessary before visualizing the bands ina gel?
So after application to a gel I see strong bands at what I believe to be 220bp. These bands only appeared in lysates that were crosslinked for 10 min, I saw no bands in lysates crosslinked for 20 min.
What I'd like to know is:
1) Do I have to purify my DNA before coming to any conclusions about sonicating efficiency?
2) What does this band at 220bp mean, and how can I change my protocol to achieve a 200-100bp smear?
I'm starting a ChIP experiment using mouse brain tissue. Briefly, fresh or frozen tissue is homogenized in cold 1xPBS, I cross link with 1% (total conc) formaldehyde for 10 and 20min at RT( some samples at 10 min, some for 20), quench crosslinking with 0.125M Gly, then incubate for 5min RT, then spin at 2k for 5 min to collect cells. I resuspend the cell pellet in 0.5ml SDS lysis buffer and incubate on ice for 10 min.
Then I sonicate using a Sonics vibracell130 (or something like that) with a 2mm probe, I sonicate at 30% amplitude. samples in a 1.5ml eppendorf are partially submersed in ice cold water during sonication. I've used several different sonicating procedures:
6 pulses for 25" with 60" rest between pulses
8 pulses, 10", 60" rest
10 pulses, 10", 60" rest
15 pulses, 10", 60" rest
No foaming occurs
After sonicating, I spin down the sonicates at 5k rpm, for 5 min, collect the supernatent then I aliquot the supe and apply some directly to a 1.5% agarose gel.
I know one should first add proteinase K, RNase, reverse crosslink, etc, but I was wondering if any of these steps are necessary before visualizing the bands ina gel?
So after application to a gel I see strong bands at what I believe to be 220bp. These bands only appeared in lysates that were crosslinked for 10 min, I saw no bands in lysates crosslinked for 20 min.
What I'd like to know is:
1) Do I have to purify my DNA before coming to any conclusions about sonicating efficiency?
2) What does this band at 220bp mean, and how can I change my protocol to achieve a 200-100bp smear?
I've never gotten interpretable results by running unpurified chromatin on agarose. We always extract the DNA first.
As for obtaining a 100-200bp smear, I don't think that is likely. Most can't get below 200bp with sonication. Are you trying to obtain mononuleosomes? If so, maybe you should try micrococcal nuclease.
So after application to a gel I see strong bands at what I believe to be 220bp. These bands only appeared in lysates that were crosslinked for 10 min, I saw no bands in lysates crosslinked for 20 min.
What I'd like to know is:
1) Do I have to purify my DNA before coming to any conclusions about sonicating efficiency?
2) What does this band at 220bp mean, and how can I change my protocol to achieve a 200-100bp smear?
Well, I'm not a pro in thuis field, but your results make me think the following:
1.) If you want to make a statement on the sonication efficiency on DNA only, you will have to purify the DNA to avoid the many many factors that will
interfere when you use whole cell lysates. These interferences may occur not only during sonication, but also when you try to measure the efficiency of DNA cleavage after sonication.
2.) You say a 220bp band only appears on the 20 min crosslink samples, but not on the 10 min crosslink samples.
This might indicate the 10 min crosslinking is not enough to actually crosslink the DNA to protein, and you get a 220bp DNA band.
In the 20 min crosslink sample the DNA might be successfully crosslinked to protein, and thus larger in size, leaving no band at 220 bp.
As mentioned in 1.), the phenomenon you are observing may also be because you are not using purified DNA,
Like I said, not guarantee for all this, they re just my immediate thoughts - hope I could help though
Sorry to keep bumping all these old threads, but I'm seeing something similar. Did you ever find an explanation?
I have been doing RNAse/proteinase on my sonicated samples, but no phenol/chloroform (the upstate kit doesn't mention in option 2 of the sonication optimization) and I always get a bright 300bp band.
Please do the reverse crosslinking, boil it for 15 can do, without reverse crosslinking, RNA can not be digested by RNAse treatment, and DNA can not be purified.