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

I used Upstate's and Active Motif's kit to IP my samples. I got reasonable level of enrichment in +Ab (RNA pol II and H4) as checked by std PCR and RT-PCR using SYBR green. I attempted to amplify the IPed DNA as indicated in Affy's protocol using a random-primed PCR before fragmenting, labeling and hybridizing it on Hu Promoter 1.0 array. However, I could not succeed in the first two attempts. I realy appreciate any tips on how much template to use in the first round of amplification (affy suggests 10 ul but they don't say the quantity). Affy's IP protocol uses about 50mil cells/IP whereas Upstate's and AM's protocol suggest 2mil cells/IP. I wonder if I am using too low template for amplification. Any suggestions?
Thanks

---

I did try the protocol with just control DNA and can't get enough either. They said u can start with using about 15ng DNA.

I am trying different protocol for the affy array!

---

Sigma's Whole Genome AMplification kit works very good on affy arrays, requires more than 1ng of starting material.

Nick

---

Thanks for the help. Another question I have is that isn't a primer concn of 200 uM for the 1st round and 100uM for the 2nd rnd amplification too much? I see a strong band of primer dimer at the bottom of my gel.

---

Hi, Nick:

Agree with you about WGA kit! It works great. But how did you fragment your amplified DNA before the hybridization to the Affy array? I am optimising the condition to use DNase I. Not sure if it is the best choice.

Thanks!

---

hi hn,

it's not necessary to fragment, WGA does it all for you and you get a smear ranging from 100-bp to 500bp

Nick

---

Hi, Nick:

I called affy, they said 100-500bp is too big for the affy array. According to their protocol, seems like the optimal size is around 100bp. The picture in their manual shows the peak size is 66nt! Have you tried the affy array without further fragmentation?

Thanks!

---

Hi guys,
I am planning to use Affy's "GeneChip WT Double-stranded DNA Terminal Labeling kit" which contains reagents for fragmentation. We routinely use Affy's kit for expression array. That might work for you.

---

Whoops! My bad,

the WGA allows you to obtain the amount of DNA required for the affy array, you will need to label it with the Affy kit, which then further fragments it as it labels the DNA for hybridisation.

Sorry for the misleading comments!

Nicik

---

Could you please specify what exactly you did?

Thanks!

Hi All,
I thought of using WGA to amplify and affy kit for labeling. However, since fragmentation with affy kit needs dUTP, I went back to affy's amplification and started playing with mg[]. The recommended mg[] is too low. I increased it to 2mM and added a couple more cycles and got an excellent amplification (see the image attached).[attachment=2509:Affy_chi...01_30_07.jpg]
Mel
[/quote]

---