Vassil

Hi Evan,

This is a strange situation...

Since you are just taking the input, then I don't think it matters how protected the chromatin (or DNA fragment) is. The sonication does not destroy the DNA (correct me if I'm wrong). All this to say that the input values should be the same.

It seems to be that the problem arises from the qPCR reaction itself somehow.

Please keep us posted about any solutions (even if it is just messing up with the loading of the sample).


Good luck!

Hi,

You need SDS in order to shear your DNA. NP-40 does not do the job. 1% SDS is usually well tolerated in terms of epitope concervation. You should be careful not to over-sonicate, though. This could destroy your epitope and heat the proteins inducing degradation (always perform in ice-cold water). Also, make sure that there are no bubbles when you sonicate, as this could also destroy your epitope due to surface tension.

1. It is possible that 1% formaldehyde may not be able to attache the co-activator to the desired site. To address this problem, you can use protein-protein crossllinkers, which have longer arms, and then perform the 1% FA crosslinking to link the complex to the DNA. Alternatively, I use 4% Paraformaldehyde for 25min at RT and it works fine for me (do NOT exceed 30min).

2. You should use 1% SDS. Do not replace this component of your buffer.


Good luck!

Vassil

Hi Joe,

If you only want to check the shearing of your DNA, the steps are correct.

I usually incubate the samples with RNase for 30min at 37oC and with Proteinase K for 30min at 37oC or 65oC, but overnight also works. One thing that you may try is to boil the samples (or put at 100oC) for 30min and then do RNase/PRotK treatment for fast decrosslinking.

You may also try the QiaQuick PCR purification kit or MinElute PCR purification kit from Qiagen to get your DNA. There is also a fairly good product from Feldan.

Good Luck!

Hi Evan,

Are sample 1 and sample 2 biological replicates, or they represent a different condition?

Just a thought here... DNA could be stuck to the wells due to incomplete de-crosslinking. The bound proteins block movement through the agarose gel. You could try running the gel for longer to see if DNA will leave the wells. Also, maybe try your samples without de-crosslinking them for comparison (as a control). DNA from non-decrosslinked samples should remain in the wells (at least to a much higher degree than decrosslinked).