Sonication Help! (View forum version)



j47027

Posted 19 April 2013 - 08:29 AM

Hi,

I am assaying sonication conditions at the moment and getting confused with the order of steps.

When I shear my chromatin what do I do next.

RNaseA treatment - 2 hours
Proteinase K - overnight*
Reverse Crosslink overnight*
Phenol Chloroform - Next day

Is that the correct order of steps?

I don't have to use Proteinase K either..correct? and *If I choose to use Proteinase K I can reverse crosslink along with Proteinase K digestion..yea??

Many thanks

Joe

pcrman

Posted 19 April 2013 - 11:30 PM

Are you doing sonication for ChIP assay? If yes, the next step is incubation with a desired antibody, then washes, elution, reverse cross-linking, digestion ...

j47027

Posted 20 April 2013 - 08:30 AM

Are you doing sonication for ChIP assay? If yes, the next step is incubation with a desired antibody, then washes, elution, reverse cross-linking, digestion ...


Yea I am..But I am only at the stage of sonication..I want to analyse the DNA size range before I go through with the ChIP..

Vassil

Posted 30 April 2013 - 05:19 PM

Hi Joe,

If you only want to check the shearing of your DNA, the steps are correct.

I usually incubate the samples with RNase for 30min at 37oC and with Proteinase K for 30min at 37oC or 65oC, but overnight also works. One thing that you may try is to boil the samples (or put at 100oC) for 30min and then do RNase/PRotK treatment for fast decrosslinking.

You may also try the QiaQuick PCR purification kit or MinElute PCR purification kit from Qiagen to get your DNA. There is also a fairly good product from Feldan.

Good Luck!