SDS Precipitation - (Sep/28/2011 )

Ok I have a major problem with my ChIP assay. After I've reversed the crosslinks and done the proteinase K step, I inactivate the proteinase K by boiling for 10 minutes.

Then I add 5 times the volume of binding buffer from a pcr/gel purification kit. However, a misty precipitate forms (which I assume might be SDS) not matter how much buffer I continue to add. The first time I did this was using a QIAGEN kit.

A friend recommended using a nucleospin clean up kit using a buffer which specifically gets rid of the SDS. I tried this and I still have some crud in my eluates and thus my PCR shows diddly squat!

Is anyone aware of this problem occurring or am I doing something ridiculously stupid? Thanks.

I think we're going to need some more info on how your ChIP protocol works before we can help............ like buffer composition and reverse cross-link method and such

chabraha on Wed Sep 28 19:56:06 2011 said:

I'm using the millipore protocol. I elute my complex in 250ul elution buffer (1% SDS, 0.1M NaHCO₃) twice for 15 minutes to end up with a combined pool of 500ul per sample. I add 20ul of 5M NaCl to reverse the crosslinks and incubate at 65 degrees overnight.

The next day I either freeze the samples at -20 or proceed. The only time it has worked for me when was when I proceeded with the protocol and did not freeze.

I then add 10ul of 0.5M EDTA, 20ul Tris-HCl pH 6.5 and 2ul of 10mg/ml proteinase K and heat at 55 degrees for 1 hour. I then inacivate the enzyme by boiling at 100 degrees for 10 minutes. I then proceed by adding 5 times the volume of binding buffer. The kit I use is this, and the binding buffer is NTB, which is mentioned at the bottom of the page: http://www.mn-net.com/tabid/1452/default.aspx

I think I may have found the problem. I tested this by adding my elution buffer plus the 5M NaCl to an eppendorf, then froze. After defrosting to RT, I added the binding buffer and the milky precipitate appeared. It appears the freezing step does something to disrupt the SDS content of the buffer.

Freezing and then thawing samples can lead to irregular distributions of salts......if you want to, try thawing the sample at ~55 degrees with shaking, this may help avoid localized buildup of salt concentrations.