Always signal in negative control :(

[pluto-nium244]

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

[zxthhr7798]

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

[zxthhr7798]

Originally Posted by pluto-nium244

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

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?

[chotie]

Originally Posted by zxthhr7798

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?

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.