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SDS Precipitation


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#1 Dave_Kub_11

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Posted 28 September 2011 - 07:38 AM

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.

#2 chabraha

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Posted 28 September 2011 - 11:56 AM

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
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#3 Dave_Kub_11

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Posted 29 September 2011 - 02:31 AM

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

#4 Dave_Kub_11

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Posted 29 September 2011 - 04:48 AM

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.


#5 chabraha

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Posted 29 September 2011 - 06:49 AM

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.
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