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
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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