Post sonication RNAse & Prot K treatment - What are the bands? (Mar/10/2008 )

Hey All,

I've been doing some sonication and following this up with RNAse & Proteinase K treatment before running on a gel. I usually run the non-RNAse/Prot K treated equivalents on the gel as well just to compare. When I do this there appears to be a band within the smear (in the non treated samples).

Can anyone suggest what this is? Is it caused by protein still crosslinked to the DNA? I would have thought this would make it move much more slowly through the gel, not right near the smear.

NOTE: Look at samples 5-8 in the photo, Lanes 1-4 obviously didn't work!

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Hi,

In my opinion, maybe a trace of RNA and some proteins.Without treatment, both of these can appear in sample.

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To me it looks as I would expect. The non-treated samples have a lot of retention within the wells or a band at the top of the smear(due to poorly migrating protein-DNA complexes).

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Thanks for the re-assurance. It was the band in the treated samples near the smear which had me curious. Surely the sonication won't create a whole bunch of very similar sized fragments?

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Can someone provide me with details on the RNAse, proteinase K treatment for checking chromatin? I've been using the Upstate kit and they suggest 2 hours at 62C with Proteinase K, followed by clean up using their columns. They also mention RNAse, but don't say what concentration or what kind.

Anyone have a catalog number for RNAse? There are a ton of them available. Concentration?

Is it okay to use a Qiagen kit for cleanup? Which one would you suggest? Any modifications required?

Here's what I'm thinking:

1) Dilute samples with 10xvolume of ChIP Elution Buffer
2) Add RNAse (how much?) and incubate at 37C for 30 minutes (?)
3) Add Proteinase K and incubate at 62 for 2 hrs
4) 95C for 10 minutes
5) Qiagen cleanup

Any suggestions?

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Hi, heres a quick guide to what I've been doing lately to check shearing.
- take 5ul of sonicated sample (5ul from a total of 400ul containing 5 million cells).
- add 5ul RNAse Cocktail (Ambion, Catalog # AM2286 or AM 2288 depending on quantity, 5ul cocktail = RNAse A - 2.5 units,
RNAse T1 - 100 units). Incubate 2 hours at 37 degrees. 2hrs may be overkill perhaps, but I like to be sure.
- add 5ul Proteinase K (not sure about concentration, but it is Proteinase K from Promega, made up as per instructions). Incubate 2
hours at 62 degrees and 10 mins at 95 degrees.
- Samples are then ready to run on a gel, so don't bother with the clean up unless you are doing a PCR.

Good luck with it!
Dave

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Thanks Dave. Do you dilute that 5ul of sample in something before adding 5ul of RNase?

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No, just 5ul of sample with 5ul RNAse. At the end I can load 5-10ul of the RNAse/Prot K treated sample on the gel and it shows up nicely.

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Hi,

just a few questions. Isn't it necessery to do a reverse crosslink before the treatment with RNase and Prot K ? Why do you incubate your DNA for 10 min at 95°C. Won't you denaturate your DNA ? Of course it will renaturate again but why this step before you put in on the gel...?

I am doing sonication with a Branson-D250 and a microtip in an 1,5 ml Eppi volume about 500 ul? Whats the best position for the microtip in the tube...

Thanx a lot

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The reversing crosslinks is done with Prot K treatment - the histones will be degraded by the Proteinase K as they are proteins. As for the 10 mins at 95, that would be to inactivate the enzyme and maybe even be done to further undo the crosslinks. I got that step from the Upstate Magna ChIP method.

I use a Branson-250 sonifier. I had much better luck using 2ml tubes that have more of a flat bottom than the 1.5mls. I use 400ul but 500ul should be fine. I found with any less than 400ul and it was too easy to foam it all up = bad. I keep the tip anywhere between a few mm's from the bottom, and make sure it is at least a few mm's from the top. Too low and it won't work properly and too high and it will foam.

Good luck!
Dave

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