ChIP assay - Foaming in sonication and other woes - (Jan/27/2005 )

I just noticed the photo of you gel pcrman,

there have been some changes to the website, and the inclusion of photos with the messages is just awesome!!!

great to see lowering the settings worked for ya! seems so simple a change doesn't it?

Nick

I have been using the Upstate ChIP kit quite a while. Here are some tips which I think are important but not documentated in the protocol.

1) When taking the agarose beads using a yellow tip, cut off its end otherwise you will end up with dry beads only left (because the diameter of the beads is roughly the same of lumen of tip end.

2) After reverse crosslinking at 65C for 4 hr and one hour of proteinase K digestion at 45C, before going to phenol/chloroform extraction, you have to transfer the samples into a new tube because the cap no longer fit tightly on the tube after intensive heating. If you don't, your sample will leak when you vortex.

Thanx for all, although I haven't take ChIP in practice....
There are many nice man tell me the tricky point in experiment...
These help me to improve my exp.
Thanks very much, Everybody...

Does anyone have a protocol for sonicating crosslinked DNA on a Fisher 100?

hi pcrman,
Can you tell me what is provided in the Upstate ChIP kit? i.e. how different/more convenient is it by using the kit than doing it from scatch?

Thanks

Hi koira,

Please check the product page which gives details what are in the kit. Actually there are not many things special except the Protein G Agarose/Salmon Sperm DNA. All buffers can be home brew.

ChIP Dilution Buffer
Low Salt Immune Complex Wash Buffer
High Salt Immune Complex Wash Buffer
LiCl Immune Complex Wash Buffer
SDS Lysis Buffer
TE Buffer
5M NaCl
1M Tris-HCl, pH 6.5
Protein G Agarose/Salmon Sperm DNA
0.5M EDTA

http://www.upstate.com/browse/productdetai...ChIP++Assay+Kit

Antibodies are not included in the kit.

Thank you pcrman.

I checked the manual of ChIP kit from both Upstate and Active Motif. As you said, the Upstate one contains buffers and PA beads, but no Abs. The Active Motif one also includes +ve ctrl ab and -ve ctrl IgG, as well as +ve and -ve primers for the +ve ctrl ab. I'm considering tring one Kit, but don't know which one is better.

I've tried ChIP on my TF, NF-Y, for a number of months already. I'm working on transient-transfected mammalian cells with promoter construct ligated to luciferase gene. And the pcr primers encompass my promoter and the luciferase sequence, hoping to show a difference with co-transfected expression vectors. The reason why I don't look at the endogenous promoter is that I want to avoid the interference from the upstream elemenets.

My problem has been a persistent band in the no-ab ctrl, sometimes stronger than the with ab IP itself. It shows up at the expected/right size no matter what primer set I use. I've read through lots of posts re:ChIP in this forum, and found some people also had problem with bands in no-ab samples. But theirs didn't seem to be so persistent. I did try to pre-clear the beads. Didn't seem to help.

Any insights/suggestions

koira,

it is not all uncommon that you see a band in your no ab control as the region you are amplifying will be present and PCR can theoretically detect one molecule.

you should see a more intense band in your AB IP though after a limiting number of rounds of PCR.

good luck!!

Nick

Hi, I am new member.
I would like to ask if there any methods to detect methylation of histones except from Chıp method.

And what can I use instead of sonicator in order to DNA fragmentation

Thank you for your advice.......