ChIP sonication problem - (Oct/17/2006 )
hi every one
i am opitimising sonication for KGN cells i use a sonics vibra cell sonicator. i tried 300ul total volume 20% with 5X, 10X, 15X, 20X for 30 sec on ice and ice for 30sec between bursts. however when i reverse crosslinked and treated with proteinase K i got a smear from 600-100bp, most brightest in 200bp area for all the samples. there were no real differences between samples although i had used increasing number of sonications. do i have to purify DNA ater crosslinking? i ran gel ithout purifying. also, is it possible to overshear the dna is so hw would i know? please help me.
The thing you are seeing is actually RNA. Unfortunately, when most papers show/claim their
sonicated "DNA" at 200 bp it is actually RNA they are seeing
To optimize your sonicated DNA you need to treat with RNAse
to make sure you get rid of this, otherwise you will be unsure if you are seeing RNA or DNA.
The problem you are encountering usually arises during phenol:chloroform extraction
and the genomic DNA gets "lost" (at the interface???).
A way to circumnavigate this issue is to run your genomic DNA after proteinase K but prior to phenol:chloroform (you can simply run a small portion of it). It will be a little messy but you will see the true size of your DNA. Try it and let us know if it works.
you must have misunderstood me. I did run my sonicated DNA after proteinase K digesrtion. I didi not do any P/C extraction prior to running on gel. but i saw a smear from 500 to 100 with the most prominent area being around the 200bp region.
please reply as soon as you can
as mikew had asked, did you treat your samples with RNAse as what you maybe seeing is RNA from your preps. it woul dbe best to send a gel image to the forum for all to see.
Run a sample that has not been sonicated.
This should give a blob of very large molecular weight.
Do a single sonication of this (~10 sec) at low intensity.
This should spread out the blob a little.
If all you see is your 100-600 bp smear when doing this,
it is definitely RNA you are seeing. You overcome this, maybe use more DNA so it will
be brighter in comparison to your RNA and most importantly do a RNAse digestion.
I had exactly the same problem as you. I had to RNase treat my samples.