Could you explain experimentally what (or how) I should do exactly in the "DNA enrichment" step during Cromatin Immunoprecipitation (ChIP) method.
In my experiments, I (think) obtained the DNA fragments which are spesific for my target transcription factors in elution buffer. To analyze them, I loaded all elution buffer containing the target DNA fragments to agarose gel for extraction. I saw some bands in one study, but I couldnt in the other. But the problem is, even I see the bands, I couldnt extract DNA from gel. So, I think, I have to tide over the loss of DNA...
Thanks a lot...
Hi Jasmin, thanks you for kindly explanation. But unfortunately, I cant go to PCR, as you tell. Because I am looking for some unknown genes sequences which are directly regulated by my target protein in Bacillus subtilis genome.
Of course we know the protein sequence, ıts gene sequence and its spesific binding site. But now, I am not interested in these sequence. I am seeking the other new unknow gene sequences related with my protein.. So I didnt dizayn primer. In fact, at first I must clone the DNA fragment which I will obtain from ChIP, and then get their sequences. To do these step, I am trying to see and extract the DNA fragment on the gel.
OK, shortly, here the case:
I set up reaction mix with DNA and protein in appropriate buffer (not in vivo), and then go on to he CHIP protocol (CROSSLINKING, STOPPING, REVERSE CROSSLINK etc..) totally in vitro. So, It will make me happy to see any gel sample showing the DNA fragments at the end of ChIP. Maybe I can compare some value for my reaction content....
I can sent you my gel picture, if you want to interpret it....
Thank a lot....
By the way, I think I had better to indicate that I dont use any commercial ChIP KIT...
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