4 replies to this topic

[Jing Wang]

hi, all
I am trying to do ChIP on chondrogenic pellets but cannot get DNA signal on agarose gel at all.  Can anybody help me?!!  What i did was i ground my pellets on dry ice and passed them through fine needles.  At this point the cells should be completely lysed because this is how I extract RNA from this kind of pellets.  Then I added nuclear lysis buffer it, sonicated DNA, and ran it on agarose gel.  And no signal at all!!!! i am pretty sure the cells should be lysed and I feel it doesn't make sense that I cannot get any DNA signal on the gel.  Even the monolayer cells had no DNA signal either!
Can anyone help with this?  thanks a million!!!!
Jing

[chabraha]

How many cells worth of DNA did you run out on the gel? Did you try PCR on a reverse cross-linked, cleaned up portion of the DNA?

[Jing Wang]

chabraha, on 24 May 2011 - 07:53 PM, said:

How many cells worth of DNA did you run out on the gel? Did you try PCR on a reverse cross-linked, cleaned up portion of the DNA?

i loaded 0.2 million cells equivalent per lane.  I haven't reverse cross-linked them yet since I feel like i lost DNA because of something.  I need to repeat this again tomorrow but for now it doesn't make sense to me at all that there was no DNA signal at all.  Do you have any idea or suggestion?  Much appreciated!!!

[chabraha]

The fact that you haven't reverse cross-linked or ProK treated the samples makes me wonder whether your chromatin is even running into the gel.........do you see an RNA smear ~100bp? You many just have to use more cells and do the reverse cross-link/cleanup.......maybe run your sample on a bioanalyzer instead since it has lower sensitivity.

[wangjing]

I sonicated my sample and loaded on agarose gel to verify the sonication.  however, the samples were all stuck in the well, didn't migrate at all.  So that means my samples are not responding to sonication at all, even I tried longer sonication.  is there anyone had similar problem?  Can anyone help me? thanks a million!!!!