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How many ug of DNA per ChIP is recommendable.

liver tissue DNA concentration ChIP

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5 replies to this topic

#1 Txaio83

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Posted 09 October 2012 - 04:13 AM

Hi! I´m trying to set up a ChIP protocol for mice liver tissue, and I wanted to know how much DNA I need to use for each IP. Is 7ug too much or too little material per IP? And 50 ul of beads would be enough for this concentration of DNA? Do you think I can trust the concentration values I get in the Nanodrop after sonicating? Otherwise, how do you calculate the amount of DNA you add per IP? You just calculate it according to the mg of tissue needed per IP? Thanks so much, I am quite lost, so I would be glad if you could give me any suggestions.

#2 vic17

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Posted 12 October 2012 - 09:07 AM

Hi, I think that depends on what you are trying to ChIP - i.e. histone markers vs. TFs. I've used around 10 ug (the lowest I've tried) with histone modification markers and it worked very well for me. Highest I've tried is 50 ug for a very difficult transcription factor. I start with very minimal tissue sample and this prevented me from trying higher concentrations. As for concentration measurements, I use the nano drop which has been going well for me. I know from the literatures, that Picogreen is a better option as it is more sensitive for this kind of experiments, but haven't tried that yet. I think as long as one method works well and consistent - stick with it! Hope this helps.

#3 Txaio83

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Posted 14 October 2012 - 11:48 PM

I am trying to ChIP some coregulators that don´t attach directly to DNA, so I guess I should use maybe more material that I was using. I am new in this and didn´t have any clue about how much material to use per IP! Posted Image now I know and can start testing! thanks a lot!

#4 Txaio83

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Posted 18 October 2012 - 07:40 AM

Sorry, I have another question. How do you quantify the DNA? I reversed the crosslinking of a small aliquot of DNA, and measured the concentration before and after purification with the Qiagen kit, and found a big difference in the concentration. I guess the value I get from the purified DNA might be more accurate, without the proteins and the buffer that can interfere. From a tissue sample of 50mg (liver) is it ok if I get like 60ug of DNA? thanks!! Posted Image

#5 vic17

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Posted 18 October 2012 - 12:33 PM

I found the same as well with the Qiagen purification kit. You can actually see the curve (if you are using a nano drop) going from stepwise to smooth and peaks at 260 indicating a more purified sample. I think 60 ug is a little low since you would typically need around 10 ug (lowest I've tried) to more than 100 ug for ChiP. I was having difficulties with a transcription factor - tried twice and it didn't work, and I came across a literature yesterday saying that they've used 150 ug for ChiP with this particular antibody! But then again if your target is abundant , it is worth giving a shot with the amount you already have. good luck and hope it works for you!

#6 Txaio83

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Posted 22 October 2012 - 07:15 AM

Great, thanks a lot. Posted Image





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