Hi Liyana,

I agree with Meng. Without amplifying your immunoprecipitated DNA (e.g. by PCR), it is not surprising that you do not see it in an agarose gel. Even when you start your experiment with millions of cells, the ammount of immunoprecipitated DNA is very small (in the picogram range) and can normally only be quantified with extremely sensitive dyes, such as picogreen.

I would recommend that you try amplifying your immunoprecipitated DNA and your negative control. In a successful ChIP you should then see a difference between the two. You can start by doing this for a locus of interest where you expect to see signal: you would just need to design specific primers for it and use a small amount of your ChIP samples in a PCR reaction.

Hope that helps a bit, and good luck!

     Diego

Hi Diego and Meng Wu, thank you for your suggestions! However, I don't know which region of DNA my protein binds to thus, I am unable to design primers for my DNA of interest. The reason I tried using Chip-seq was to find out which region my protein binds to.. I've nanodropped my samples and they turn out to be 3.1-6.5 ng/ml. but the quality of DNA in each samples are quite good with a ratio of 1.8 average for each samples. 

I think your ideas about my DNA concentration being extremely low are correct.. now I'm trying to find out how I can validate if there are DNA available for sequencing..

Hi Liyana,

Meng Wu and Diego are both right, and give good hints. At the concentration that you're measuring, the Nanodrop is not accurate anymore. You will therefore need a more sensitive machine. The Agilent Bioanalyzer is your best choice, as it will also give you information about quantity and quality. A simpler option is the Qubit (Life Technologies), that will tell you the amount of double stranded DNA.

If the aforementioned machines tell you that DNA is available, a simple PCR/qPCR should tell you if there is bacterial DNA present. Considering that ChIP experiments generally have quite a strong background level of DNA, combined with the small size of bacterial genomes, you should have no problem to get a product using any good primer set.

This, naturally, does not tell you if your ChIP experiment has enriched for binding sites in the genome. Without any other leads, you will not be able to find this out though.

Hi Liyana

Pico green is a great and accurate way of measuring very low DNA concentrations. I would also like to suggest PCI purification of ChIP enriched DNA using Phase lock tubes (heavy) as all column based purification techniques will lead to significant loss of material. PCI purification with followed by glycogen assisted precipitation via O/N -80C incubation takes longer than column purification but gives close to 100% recovery and better quality DNA in my experience. We can detect even very low affinity binding events with high statistical probability in the final ChIP-seq data using this approach.

That said, doing a "blind" ChIp-seq experiment that you are doing is tricky. Once you have identified a set of novel binding sites, it is therefore imperative to design both positive and negative qPCR primer pairs and perform an extensive series of conventional CHIP-qPCRs to verify the accuracy of you result.

Best of luck,

Patrik

Hi Liyana,

I have one suggestion to find some target sequences you could later use for validation PCR for your library - why don't you transform the IP'ed DNA into a cloning vector for sequencing (usually even very small amounts should work fine),  then transfrom competent bacteria, and isolate several colonies, make mini-prep cultures, isolate the plasmid, confirm that you have insert by restriction digest, and perform Sanger sequencing of each plasmid - it may give you a rough direction what kind of genes/DNA your protein binds to.

Another question, I guess the antibody you use is not validated for ChiP-Seq, (if yes, you can ask the provider for some target genes), so before going into sequencing, I would test if it recognizes the protein of interest in fixed cells.