I am using HEK293 cell line and following this protocol from Abcam:
https://www.abcam.com/en-us/technical-resources/protocols/cross-linking-chip-seq?srsltid=AfmBOorG7SIwO1fUpZFjCRRSqZpupubCLv4zHK4t6gCQCZ5U9I0ndxcu
Up to sonication, my protocol is optimised, and i am getting fragments between 200-500 bp (image attached),
After sonication, I took 100 ul of the sonicated samples and did immunoprecipitation. i used 5ug of Cl8Wg16 antibody against RNA Pol II [it is a test experiment so used to most common possible]
In the end, I used Qiagen PCR purification kit and eluted immunoprecipitated DNA in 30 ul of EB. For samples, i am getting "DNA too low to be detected" and for 2% input control i am getting 5-6 ng/ul of DNA.
If you know ChIP and don't help me, I will really cry now!!
please email me [email protected] or reply to this post or message on instagram @pradip_the_great.
thank you in advance,
-Pradip
It is not uncommon to get a very low/undetectable yield after ChIP. It does not mean it failed, and the quantity (in ng) of DNA is not proportional to the quality of your sample (at least it's not in my hands...).
To know if your ChIP has worked, you need to check by qPCR the enrichment of a region you know your target binds to versus a region you know it does not bind (signal over ratio). It is also a good idea, since you are setuping up the experiment, to add a negative control (Igg antibody).
Also, I've never heard of using PCR purification columns to extract DNA at the end of the experiment, you might want to perform a regular phenol extraction instead.
Good luck !
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