ChIP on chip - can anyone help me? - (Feb/04/2008 )

Hi everyone!!!

After the very positive repsonse and help I received last time I posted about my ChIP problems, I was hoping that I could get just a little bit more help with ChIP on chip:)

So, I seem to be doing ok with my ChIPs, am using the fast ChIP method now and I want to begin ChIP on chip. I plan to use the Sigma kit WGA2 and am just wondering about how much DNA I need to amplify. I typically get about 10ng from my ChIP samples. The kit suggests using 1ng/ul - is this the typical amount one needs before amplification? How much DNA do you require for ChIP on chip?

Any suggestions would be greatly received!

Thanks for your time

H

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How many cells are you using per IP? I am using less than 1 x 10^7 cells/IP and I use the Roche micro kit to purify my Chip samples. I elute my Chip samples in 13ul (my input in 20ul). I use 1ul of my Chip for qPCR and use 10ul for amplification. I don't nanodrop my IP samples so I don't waste any DNA. I amplify 10ng of my input DNA. I then use 2ug DNA to label my samples with the invitrogen Bioprime kit and this gives me enough DNA for ChIP-Chip. Hope this helps

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Hi Clare,

Thanks for your response. I use about the same number of cells as you do and I seem to find when I purify my DNA, my PCRs don't look as nice as when I use the crude DNA from the ChIP. In my previous method though, I did purify my DNA before PCR with a clean up kit. Do you use the Whole Genome Amplification method to amplify your DNA?

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If you are using Fast ChIP you probably should use a clean-up before doing the amplification. We didn't include this in Fast ChIP because the DNA was good enough for real time PCR (which is what we do almost exclusively) but I know that other labs that have used it (Brad Bernstein) needed to add a column cleanup step before the amplification and/or hybridization.

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Hi Clare,

Thanks for your response. I use about the same number of cells as you do and I seem to find when I purify my DNA, my PCRs don't look as nice as when I use the crude DNA from the ChIP. In my previous method though, I did purify my DNA before PCR with a clean up kit. Do you use the Whole Genome Amplification method to amplify your DNA?
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Yes, I am using the WGA kit

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