Hi all- my first post, so please be kind if I miss some expected content.
Why doesn't my post amplification enrichments match my pre amplification enrichments?
To check the quality of my recently generated libraries from ChIP, I performed qPCR on positive and negative control loci. However, the levels of enrichment no longer look like what they did on the unamplified/pre library DNA. For some the trend is correct, but not for others.
This was a well validated ChIP for histone marks.
Fragment size of IP-DNA ~200bp and library ~300bp confirmed via Bioanalyzer.
I made a library for both the input and the IP-DNA.
Pre library- I used equal vol of DNA per qPCR reaction.
Post library- I used equal concentrations of DNA per qPCR reaction.
quantification via Qubit.
Any thoughts would be appreciated. I'm not sure if people normally do this even. Thanks!
Why doesn't my post amplification enrichments match my pre amplification enrichments?
To check the quality of my recently generated libraries from ChIP, I performed qPCR on positive and negative control loci. However, the levels of enrichment no longer look like what they did on the unamplified/pre library DNA. For some the trend is correct, but not for others.
This was a well validated ChIP for histone marks.
Fragment size of IP-DNA ~200bp and library ~300bp confirmed via Bioanalyzer.
I made a library for both the input and the IP-DNA.
Pre library- I used equal vol of DNA per qPCR reaction.
Post library- I used equal concentrations of DNA per qPCR reaction.
quantification via Qubit.
Any thoughts would be appreciated. I'm not sure if people normally do this even. Thanks!