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ChIP elution from Dynabeads and evaporation

ChIP

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#1 Hic5Prostate

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Posted 24 March 2012 - 07:04 PM

I need some help. I am performing a ChIP on my samples and got all the way to the elution step. Since I use Dynabeads, I added Proteinase K to the elution buffer and incubated my tubes overnight on a heated shaker (65 C, 1000 RPM). When I came back today, I noticed that two of my tubes were nearly desiccated, but with a decent amount (~50 uL) of buffer remaining. I diluted these tubes with 200 uL of elution buffer, isolated the supernatant, and proceeded to phenol/chloroform/isoamyl alcohol separation. Upon reading online that evaporation could hamper cross-linking, I took my beads and re-incubated them in 400 uL elution buffer and 5 uL Proteinase K. However, now I'm concerned that since the Proteinase K was added right after adding the elution buffer (which is what my protocol says to do), the antibodies would have been cleaved, and there would be nothing left on the beads. Which sample is more likely to have my sample: the initial eluate, the second eluate, or did the evaporation ruin my experiment?

For the record, the postdoc in the lab, who has extensive experience with ChIP, says that evaporation shouldn't damage the DNA (supporting my feeling that a 200 uL dilution/wash of the tube should do the job just fine) and that drying DNA in a speedvac won't damage it.

#2 Hic5Prostate

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Posted 25 March 2012 - 11:18 PM

I think things turned out fine. The eluates from the first overnight incubation (the one with the evaporation) produced pellets, and the second set of eluates also produced pellets. I ended up pooling the two together, so it's as if the evaporation never happened. I guess the Proteinase K addition was the key to successfully reversing the crosslinks in the face of evaporation, in case anyone who has the same problem stumbles upon this thread months or years from now.





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