Always signal in negative control :(
 

I have a big problem...

I use the ChIP for yeast cells and my target proteins should binds on a distinct DNA-regions...

My Problem is, that in every experiment i´ve done, after a normal PCR my negative control has the same band (same intensity) as my positive control..

I use magnetic dynabeads from Invitrogen (IgG2, Sheep-Anti-Mouse) and my protein is tagged with a HA3-Epitop and is use Mouse antibody...

In the samples without antibody is in the majority of cases no signal...

But in the negative control (I use gene-primers, on which my targets proteins dont bind; in theory) is always a signal :/

I tried to preclear the samples with BSA, salmon sperm DNA und glycogen...without success....

I use a lot of chromatin samples and very little...also without success...

I don´t know, what i should do...

Have anybody any idea?

Also to reduce the number of cycles of the PCR were without success...

Greetz

Re: Always signal in negative control :(
 

DO you slove your problem ,and I meet the same question . If you sloved it ,could you tell me what you do? thank you .:cry:

Re: Always signal in negative control :(
 

I have the same question, and in the samples without antibody have signal as much as with antibody ,could you tell me the protocol you do?

Re: Always signal in negative control :(
 

So do I, I've got band signal when I perform PCR on mock experiment which I don't add antibody and protein agarose bead, also on using IgG. Their bands are as much as in experiment with antibody added.

At first, I think maybe my some of my reagents in ChIP is contaminated, when I detect I found that they are not. I don't know how to solve my problem. :(