I am doing ChIP for MeCP2 from total brain Chromatin. I start with 25ug of Chromatin. Assuming my antibody works good, how much DNA do you usually recover from your ChIPs in terms of ng/ul?
thanks
Gilda
Starting with 1 million cells I've obtained values between 0.3-0.8 ng/ul in around 40ul
You must be using 5 to 10 million cells per ChIP. at the end considering that you are using a good antibody you should easily get in the order of about 20 to 40 ng total yield. However if you are using salmon sperm DNA to equilibrate your beads that is going to skew your result.
I usually start with a lot more material (500 ug) and end up obtaining anywhere from 10-200 ng/ul of ChIP'd DNA.
The amount of recovered DNA hugely depends on the abundance of your mark. You need to check the recovered percentage from an experiment using the same antibody on the same tissue.
I start ChIP experiment with 25ug of chromatin and obtain 10-30ng/ul of gDNA diluted in 40ul depending on the antibody and how abundant your protein is, if it's not very abundant I increase amount of chromatin upto 50ug. However There should be plenty of Mecp2 in brain.
This varies quite a bit between cell types (i.e. abundance of the mark or protein in that cell type), the antibody and the nature of the protein itself (histones usually pull down more DNA than many TFs). For an IP for a secondary binder from 20m B cells, I've got between 5-8ng DNA in total. This is in 40ul elution buffer, so your DNA measurement can seem low - eg 125pg/ul. Although, if you're doing Illumina Truseq library prep, 5ng DNA should work - just be sure to have validated known protein binding targets and negative controls (regions where MeCP2 definitely doesn't bind) by qPCR on the DNA first.
As Stuti has mentioned that ChIP DNA yield depends on various factors such as cell type in question, abundant of the mark or protein (histone have high binding coverage than TFs and the antibody quality. For an IP for histones using 20ug of chromatin DNA from T cells as starting material I have got between 15-50ng DNA in total. For TFs I usually got 5-25ng from 25 million cells (200ug chromatin).
I hope this will give you an estimation.
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