I am performing magnify chromatin immunoprecipitation on thalamic tissue samples to determine if the primers we have for VGAT work via q-PCR.

Before I ask my question, I want to put out there that this is the first time I am performing this and our first run did not end with a very pure DNA sample.

My question pertains to the shearing of the chromatin (that is in the protocol that is attached and starts on page 24). I do not have access to the lovely, convenient automatic sonicators. All I have access to is an analog Branson 450 handheld sonicator probe (Well, it is on a stand so technically, I do not hold it).

The protocol has different examples for using a Bioruptor or Covaris sonicator but not for the handheld probe sonicators.

What is a good starting protocol for using a handheld sonicator on brain plugs (about 2mm punches but I use 4 together in one tube)? I assume the sample needs to be on ice while sonicating but how long does the sample need to sonicated and how many times should this be done (example, sonicate the sample on ice for 15 seconds on and then a 15 second break and repeat this 5 times). I believe we need 200-500 bp fragments. I know this is all individualized but having a starting point would help and give me something to adjust and fine tune.

Does anyone have any suggestions or recommendations for timing with the handheld sonicator probe for shearing chromatin (from a 2mm brain punch) into 200-500 bp fragments?