4 replies to this topic

[Dave_Kub_11]

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.

[chabraha]

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

[Dave_Kub_11]

chabraha, on 28 September 2011 - 11:56 AM, said:

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

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.co...52/default.aspx

[Dave_Kub_11]

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.

Edited by Dave_Kub_11, 29 September 2011 - 04:49 AM.

[chabraha]

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.