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Old 11-25-2009, 03:41 AM   #1
aleidenroth
Junior Member
 
Location: Nottingham, UK

Join Date: Nov 2009
Posts: 8
Default Covaris S2: Chromatin shearing for ChIP(-seq?)

Hello everyone,

I'm new to this forum and have just started a PhD.

I'm planning to ChIP an epitope tagged protein (a transcription factor I'm expressing exogenously in transient transfections).

Currently, I'm testing shearing conditions on the Covaris S2 - has anyone used it to shear chromatin for ChIP applications? The lab who owns it here just uses it for Deep-SEQ library preps.

I can get the DNA into a size range of 200-700 quite happily, although the sample gets very warm during sonication. Here are the conditions that I use:

Sonication volume: 500ul

Treatment 1:
Duty Cycle: 20%
Intensity: 8
Cycles per burst: 200
Cycle time: 60 seconds

Treatment 2:
Rest (to prevent warming)
30 seconds

Cycles: 20

Tube gets warm even after only one Treatment 1 60 second exposure. Will this affect protein integrity or cross-linking, and if so, should I try and drop the sonication time to 30 or even 15 seconds? How about dropping the intensity?

Also, what size range is suitable for downstream ChIP-seq? Depending on where I look, suggested size ranges vary quite a bit.


Thanks in advance for any feedback / nuggets of wisdom.

Andy
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