Dear Haque,

I followed the following link where good suggestions are given.

You need to normalize your sample as suggested in the following link. Check if this is useful for you.

http://www.protocol-online.org/biology-forums/posts/29733.html

In general, when you preform ChIP-qPCR and then calculate %input, it will be less than 100%, meaning there is less of your DNA in the IP than in your input. 

In your input sample you have all the copies of that DNA sequence (number of cells *2) while in your IP sample you have only the copies that were bound by your protein (1 allele and less than 100% occupancy) and were IP-ed (which is never 100% efficient). 

I know that for histone modifications you should expect less than 10% %input for a good ChIP. For transcription factors even less.

Than being said, you should remember to normalize your results since you've taken different amounts of DNA for input and IP.

In agreement with the Nitsan, I would like to mention that the iput DNA (20uL) is much more concentrated than the IP samples. IP makes a so called filtration and hence, if you are looking for a moeity interaction, a concentrated DNA would have a faster Cq (lower Cq) than filtered diluted strands. Are you looking for any sort of Protein DNA interaction?

Best,

Somnath.

Generally, 1% of the starting chromatin sample is used as input. Usually it does not give lower Cq than your IPed sample in qPCR. As Nitsan said above, after qPCR you need to normalize your results as %input.

Adjusted input to 100% is [Cq Input - 6.644] where 6.644 is  log2 of 100 as 100 is your dilution factor if using 1% input.

Percent input fraction of your IP is [100multiplied by 2^(Adjusted input - Cq (IP)]

In your case you have taken about 3 % input. Normalize accordingly.

Thank you Paul for your nice comment, yes I am sorting protein DNA interaction.

M. Sarkar, thank you very much for adding your nice suggestion. This will be very helpful for me.