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Sonicator parameters for chromatin fragmentation in ChIP?
I would like to perform a chromatin immunoprecipitation experiment on plant material (different then Arabidopsis) and to be honest - I have no experience in this kind of techniques. I know that ChIP is so challenging and sonication is one of the crucial points in this procedure. What are the most important parameters of sonication in case of ChIP procedure: amplitude, frequency or probe volume? I have found protocols in which tips for volumes up to 1 ml were used to sonicate probes of about 500 ul - is it possible? I will be grateful if you would shed a light on this ‘parameters problem’.
Re: Sonicator parameters for chromatin fragmentation in ChIP?
First of all, you need to know that it is really different which sonicator brand and model you are using. For example, sonicate the chromatin solution for 10 sec, four times on 5% power using a Branson Sonifier 250, to shear DNA to approximately 0.5–2 kb DNA fragments.
So, it would be better if you try to sonicate several times with different times. I would recommend to stablish one amplitude (the most used is 10%) and then try different sonicated times (for example, 5'' / 10'' / 20'' ON, 3 times). After that, run an agarose gel and check sonication efficiency. You can decide depending on the size of DNA fragments that you will need.
As far as I know, if you increase the amplitude you should decrease the sonicated time.
I hope it has been helpful!
|The Following User Says Thank You to pluja For This Useful Post:|
ChIP Kick (12-13-2012)
|chip , chromatin , fragmentation , parameters , sonicator|
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