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Old 07-09-2014, 11:20 AM   #1
samhh11
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Location: Maryland

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Default Whole cell lysate vs. nuclear lysate for ChIP-seq sample prep (sonication step).

Hello everyone,
I'm a newbie in ChIP-seq and I'm having trouble in sample prepartion, particularly sonication step. I use the Bioruptor.
I'm just getting a huge smear when I run my sonicated samples in a gel.
With numerous tries, I ruled out over-fixation, insufficient number of sonication cycles, tubes, etc.
So the only possible factor that comes to my mind is the use of whole cell lysate.
I know that general protocols make you isolate the nuclei and use the nuclear lysate for sonication.
But I've been using the whole cell lysate including everything for sonication (using RIPA buffer for cell lysis).

Does anyone know if this could result in incomplete sonication?
I've been stuck at this step too long and I'm hoping to get some feedback to solve this
Thanks!
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Old 07-09-2014, 05:58 PM   #2
HSV-1
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Hi,
I had the same trouble for long but now I solved the problem.
In short the whole cell lysate is no problem.
You thought your samples were not sonicated well according to your Agarose electrophoresis. How did you purify your DNA? If precipitation with glycogen I think you will always get a huge smear!
To make sure your sonication is no problem you could directly load your sonicated chromatin (yes, w/o reverse crosslink , w/0 spinning down, lets say 5ul) on a 2% Agarose. You might realize that the band will be higher than the sonicated DNA. But don't worry they are very similar. If you want to see sonicated DNA quality you should purify DNA with Qiagen PCR purification column post reverse crosslink.
Do you use the steel probe to do sonication? If yes, make sure the volume is around 2ml. 12-15min will make good sonication for many cell lines


Cheers,


Good luck!
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Old 07-10-2014, 07:32 AM   #3
samhh11
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Hi HSV-1,
Thank you so much for your reply.
I've been using glycogen to aid precipitation, so I guess that might have caused the huge smear!
I use bath sonicator, the Diagenode Bioruptor.
When you use Qiaquick PCR purification column, do you follow their PCR purification protocol? (such as adding PB buffer 5 times the volume of your sample before loading to column, etc.).
Or do you just load your reverse crosslinked sample straight to the column? followed by wash buffer.

Thanks a lot!
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Old 07-10-2014, 07:03 PM   #4
HSV-1
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Quote:
Originally Posted by samhh11 View Post
Hi HSV-1,
Thank you so much for your reply.
I've been using glycogen to aid precipitation, so I guess that might have caused the huge smear!
I use bath sonicator, the Diagenode Bioruptor.
When you use Qiaquick PCR purification column, do you follow their PCR purification protocol? (such as adding PB buffer 5 times the volume of your sample before loading to column, etc.).
Or do you just load your reverse crosslinked sample straight to the column? followed by wash buffer.

Thanks a lot!
yes, treat as the reverse crosslinked chromatin as PCR.
good good luck
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