Hello community,
This is my first post, but I've been a reader of this forum for a few years now, it's a great resource, so thanks to all who contribute.
We are doing ChIP-seq for a number of sequence-specific TFs.
DNA yields are generally acceptable (25 ng, by pico-green), except for one TF for which we get at best 1 ng per IP.
We've tried a lot to optimize the IP conditions (e.g. amount of antibody, amount of chromatin, buffer composition, wash conditions, etc), but to no avail.
The IP does work in the sense that in gene-specific PCR, we do see an excellent enrichment at a few known target loci.
We've thought of performing a large number of IPs and pooling all of them together, but we are limited by the amount of antibody and chromatin we have left.
For a situation like ours, has anyone thought of mixing their low amount of IP'ed DNA with some amount of input DNA, to reach a total amount of DNA acceptable for library preparation? My rationale is that if the IP worked really well (it seems like it did), then it will not suffer much from being "spiked" with some input DNA.
What do you experts think? I searched this forum and pubmed/google scholar but did not find anything about the feasibility of this approach.
Thanks in advance for your help.
Alex
This is my first post, but I've been a reader of this forum for a few years now, it's a great resource, so thanks to all who contribute.
We are doing ChIP-seq for a number of sequence-specific TFs.
DNA yields are generally acceptable (25 ng, by pico-green), except for one TF for which we get at best 1 ng per IP.
We've tried a lot to optimize the IP conditions (e.g. amount of antibody, amount of chromatin, buffer composition, wash conditions, etc), but to no avail.
The IP does work in the sense that in gene-specific PCR, we do see an excellent enrichment at a few known target loci.
We've thought of performing a large number of IPs and pooling all of them together, but we are limited by the amount of antibody and chromatin we have left.
For a situation like ours, has anyone thought of mixing their low amount of IP'ed DNA with some amount of input DNA, to reach a total amount of DNA acceptable for library preparation? My rationale is that if the IP worked really well (it seems like it did), then it will not suffer much from being "spiked" with some input DNA.
What do you experts think? I searched this forum and pubmed/google scholar but did not find anything about the feasibility of this approach.
Thanks in advance for your help.
Alex
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