Hello,everyone, I'm doing ChIP-Seq on fruit fly embryos now, the biggest problem I got here is that DNA after IP is too little for sequencing, then I increased the mass fo embryo to 1g for one sample, still not work, both concentration and the amount of DNA are not enough, the protein I aimed at is not histone or transcription factors, so it may not associate with the chromatin so tight like them.
Now, except for increase the original amount of embryo, I can't think of other good ways, so are there any advices for it?
Thank you so much!
Maybe you could try a dual crosslinking! Quite easy to perform….Have a look to this paper…various groups have had some success ChIPping some factors
Hello,
How much of IP'd DNA do you get? How do you prepare chromatin ( one-step lysis, 2-step lysis? how do you perform washes after IP (numers of steps, buffers used), what kind of bead do you use? HOw your chromatin looks before IP?
All these steps can be optimized to get better recovery.
Hi, lexi. For me, the fixation, IP process are critical to get enough DNA. Did you detect the input DNA by a agarose gel? If you get enough input then the fixation step is work. Then try to check the quality of antibody, the component of IP buffer, even the step of reverse cross link. If any of the steps are good, then try to use more initiate tissue ( 5g to 4g maybe get enough DNA for sequencing).
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