ChIP on Affy's Human Promoter 1.0R array - (Jan/17/2007 )

Hi Mel,
I also want to use the affy protocol for amplification. Could you please explain me what exactly you mean with mg[]?

Thanks,
Fridtjof

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The Affy protocol (page 26 of Chip protocol) recommends very low mg concentration (0.75mM final, 3 ul of 25mM in 100 ul rxn vol.). I increased this to 2.0mM final, i.e. I added 4 ul of 50mM MgCl2 in the second round of amplification. I also modified the cycle conditions on p.27 to 18 cycles each. This should work perfectly. Let me know if you have further questions.
Mel

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Thank you for your information. Indeed I have further questions, and as I have just started with chip-chip there will be more to come.
I wonder about the amount of starting material I should use for the amplification. The concentration of the ChIPed DNA I have is very low, so I would like to use as little as possible DNA for amplification, less then the 2-4ng suggested by Affymetrix. Which method do you use for quantification? Which starting amount do you use?

I also wonder if increasing [mg] might lead to artefacts due to mispriming. Have you already hybridised arrays?

I really appreciate your support,

Fridtjof

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Regarding mispriming, I did not bother because it is random priming anyways. The Affy tech person also suggested to me to increase the [Mg].

I used 10ul of IPed DNA as suggested in the protocol assuming that I have enough template, and it did work well. I used Upstates kit for chipping. You may use WGA (sigma) to amplify your sample (perhaps additional artefact) as a last resort but I have not used it myself.

Yes I did hybridization and got good results. I am still analyzing the data using different tools to detect chip regions.

Hope this helps
Mel

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Mel:

How do you know the results are good or not? I just run some test samples and trying to figure out how to use the software. How do you know if your hybrdization is good, seems like there are no quality controls to look at.

Thanks a lot!

hong

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You are right. I could not do the standard QC analysis. What i did was just visual inspection of the image. I am still in the learning process. So if you come across some tips, please let me know.

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You are right. I could not do the standard QC analysis. What i did was just visual inspection of the image. I am still in the learning process. So if you come across some tips, please let me know.
[/quote]

Same thing here, it is very tough to figure out everything by myself. Especially I used WGA kit to amplify and DNaseI to fragment the DNA.

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Hi collegues,

Thank you for the help so far, I was able to get a good amplification and the enrichment of my positive targets stayed the same after amplification.
Now I have to decided to use mock IP or total input as control samples for my hybridizations. What did you use, do you know any literature what might be the better choice?
I also wonder about he number of replicates I should make.
Again, I am grateful for any help,

best wishes

Fridtjof

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Hi all,

If somebody has been able to fragment the samples successfully please let me know. I amplify chip DNA by LMPCR and get a nice smear...but when i am trying to fragment this using DNase I from Amersham, i am able to see only 20-30 bp.............

Please let me know if anybody has done any fragmentation with DNase I:

thank you.

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just try to optimise with less DNaseI and shorter period, like 5 or 10min.

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