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Proteinase K after Sonication


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#1 Firebird01

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Posted 10 June 2009 - 12:41 AM

Hi,

I've got a question concerning the decrosslinking after sonication. At the moment I'm trying to find the correct sonication conditions for my cells (lymphoblastoid cells). I take a sample after each sonication step and want to put this samples on a testgel. Is it essential to treat the samples with Proteinase K oder RNase before I put them on a testgel?

thanks for advice

#2 KPDE

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Posted 10 June 2009 - 09:26 AM

Hi,

I've got a question concerning the decrosslinking after sonication. At the moment I'm trying to find the correct sonication conditions for my cells (lymphoblastoid cells). I take a sample after each sonication step and want to put this samples on a testgel. Is it essential to treat the samples with Proteinase K oder RNase before I put them on a testgel?

thanks for advice


You should proteinase K treat and do the reversal of crosslinking before running on the gel. You don't have to RNase treat but if you don't then you'll see a fairly bright RNA smear in the 100-200bp region on your gel.

If you want to do a fast reversal of crosslinking just dilute your chromatin (10-20ul) in 100ul of 10% chelex-100 suspension. Add proteinase K and digest for 15 minutes at 50-55C and then boil or heat on a heat block at 100C for 10minutes. Then run the supernatant on the gel. This works for ChIP too. Just add the 100ul chelex suspension to your protein A beads after the last wash and proceed as above. Works great for either agarose or magnetic beads.

#3 Firebird01

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Posted 14 June 2009 - 11:54 PM

Thanks for the answer, but why is it so essential to do this before elektrophoresis?

cu

#4 KPDE

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Posted 18 June 2009 - 12:10 PM

Thanks for the answer, but why is it so essential to do this before elektrophoresis?

cu


Chromatin complex movement through the gel is severely retarded by the bulk of all the nucleosomes. A large amount of the chromatin won't even run into the gel and just remains in the well. It is impossible to determine fragment size unless you are dealing with naked DNA.

#5 Firebird01

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Posted 23 June 2009 - 01:49 AM

Thanks for the answer, but why is it so essential to do this before elektrophoresis?

cu


Chromatin complex movement through the gel is severely retarded by the bulk of all the nucleosomes. A large amount of the chromatin won't even run into the gel and just remains in the well. It is impossible to determine fragment size unless you are dealing with naked DNA.


So I think it's only necessary to reverse the PFA Fixation by heat. Because non fixated genomic DNA, which is cutted by enzymes, is able to move through the gel.
In my case, I only want to have a before and after picture of the sheared chromatin. I don't want to use the sample for further investigation like PCR.

What do you think of this protocol to reverse the crosslinking: 140 μl TE, 5 μl 5 M NaCl and 5 μl 20% SDS to 10 μl Sample.
I'm looking for a very cheap method to reverse the crosslinking.

Edited by Firebird01, 23 June 2009 - 01:52 AM.


#6 KPDE

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Posted 23 June 2009 - 11:25 AM

Thanks for the answer, but why is it so essential to do this before elektrophoresis?

cu


Chromatin complex movement through the gel is severely retarded by the bulk of all the nucleosomes. A large amount of the chromatin won't even run into the gel and just remains in the well. It is impossible to determine fragment size unless you are dealing with naked DNA.


So I think it's only necessary to reverse the PFA Fixation by heat. Because non fixated genomic DNA, which is cutted by enzymes, is able to move through the gel.
In my case, I only want to have a before and after picture of the sheared chromatin. I don't want to use the sample for further investigation like PCR.

What do you think of this protocol to reverse the crosslinking: 140 μl TE, 5 μl 5 M NaCl and 5 μl 20% SDS to 10 μl Sample.
I'm looking for a very cheap method to reverse the crosslinking.


If you're looking for cheap, as in buying no new reagents, then the traditional method of crosslink reversal (65C for 4-6 hours in a basic buffer) is the way to go. If you're looking for fast, the method I mentioned above using chelex is the way to go.

As for whether you'll need proteinase K just to run the chromatin on a gel, I'm guessing that the histones will still be bound to the DNA, even after crosslink reversal. But it's a very simple and quick experiment to find out if it's needed or not so give it a shot.

#7 Firebird01

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Posted 24 June 2009 - 06:28 AM

Thanks for the answer, but why is it so essential to do this before elektrophoresis?

cu


Chromatin complex movement through the gel is severely retarded by the bulk of all the nucleosomes. A large amount of the chromatin won't even run into the gel and just remains in the well. It is impossible to determine fragment size unless you are dealing with naked DNA.


So I think it's only necessary to reverse the PFA Fixation by heat. Because non fixated genomic DNA, which is cutted by enzymes, is able to move through the gel.
In my case, I only want to have a before and after picture of the sheared chromatin. I don't want to use the sample for further investigation like PCR.

What do you think of this protocol to reverse the crosslinking: 140 μl TE, 5 μl 5 M NaCl and 5 μl 20% SDS to 10 μl Sample.
I'm looking for a very cheap method to reverse the crosslinking.


If you're looking for cheap, as in buying no new reagents, then the traditional method of crosslink reversal (65C for 4-6 hours in a basic buffer) is the way to go. If you're looking for fast, the method I mentioned above using chelex is the way to go.

As for whether you'll need proteinase K just to run the chromatin on a gel, I'm guessing that the histones will still be bound to the DNA, even after crosslink reversal. But it's a very simple and quick experiment to find out if it's needed or not so give it a shot.


Thank you for the help, there is a big difference between crosslinking and reverse crosslinking on a gel.

#8 Firebird01

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Posted 25 June 2009 - 01:45 AM

I have got a additional question. I compared crosslinked and reversed crosslinked samples on a gel. I sonicated the cells with 50% amplitude. The crosslinked samples show a good fragmentation and you can the the fragmentation is better from one sonication step to the other. But when I reverse crosslinking of the same samples, I get a band at 200-300 in every sample (all samples look the same). Further more the unsonicated sample looks also like this. This also happens at 70 or 20 % amplitude.

Thanks for the answer, but why is it so essential to do this before elektrophoresis?

cu


Chromatin complex movement through the gel is severely retarded by the bulk of all the nucleosomes. A large amount of the chromatin won't even run into the gel and just remains in the well. It is impossible to determine fragment size unless you are dealing with naked DNA.


So I think it's only necessary to reverse the PFA Fixation by heat. Because non fixated genomic DNA, which is cutted by enzymes, is able to move through the gel.
In my case, I only want to have a before and after picture of the sheared chromatin. I don't want to use the sample for further investigation like PCR.

What do you think of this protocol to reverse the crosslinking: 140 μl TE, 5 μl 5 M NaCl and 5 μl 20% SDS to 10 μl Sample.
I'm looking for a very cheap method to reverse the crosslinking.


If you're looking for cheap, as in buying no new reagents, then the traditional method of crosslink reversal (65C for 4-6 hours in a basic buffer) is the way to go. If you're looking for fast, the method I mentioned above using chelex is the way to go.

As for whether you'll need proteinase K just to run the chromatin on a gel, I'm guessing that the histones will still be bound to the DNA, even after crosslink reversal. But it's a very simple and quick experiment to find out if it's needed or not so give it a shot.


Thank you for the help, there is a big difference between crosslinking and reverse crosslinking on a gel.



#9 zyka

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Posted 16 July 2009 - 11:49 AM

Hi guys,

My question might sound silly but... Why do you put proteinase K in your samples at the sonication step? Is it because you're working with cells? We are sonicating genomic DNA extracted from blood, do we need to add proteinase K as well??

Thanks!

#10 KPDE

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Posted 16 July 2009 - 01:09 PM

Hi guys,

My question might sound silly but... Why do you put proteinase K in your samples at the sonication step? Is it because you're working with cells? We are sonicating genomic DNA extracted from blood, do we need to add proteinase K as well??

Thanks!


If you're planning on using the chromatin for ChIP you don't use the proteinase K until after you've run the IP. You definitely want as little proteolysis as possible before and during the IP.

#11 Clare

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Posted 17 July 2009 - 12:58 AM

Hi KPDE,

As we up going to increase our ChIP numbers soon I thought I would try your fast reversal method. Who do you buy your Chelex from?
Thanks!
Clare


You should proteinase K treat and do the reversal of crosslinking before running on the gel. You don't have to RNase treat but if you don't then you'll see a fairly bright RNA smear in the 100-200bp region on your gel.

If you want to do a fast reversal of crosslinking just dilute your chromatin (10-20ul) in 100ul of 10% chelex-100 suspension. Add proteinase K and digest for 15 minutes at 50-55C and then boil or heat on a heat block at 100C for 10minutes. Then run the supernatant on the gel. This works for ChIP too. Just add the 100ul chelex suspension to your protein A beads after the last wash and proceed as above. Works great for either agarose or magnetic beads.
[/quote]

#12 zyka

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Posted 17 July 2009 - 10:18 AM

Hi guys,

My question might sound silly but... Why do you put proteinase K in your samples at the sonication step? Is it because you're working with cells? We are sonicating genomic DNA extracted from blood, do we need to add proteinase K as well??

Thanks!


If you're planning on using the chromatin for ChIP you don't use the proteinase K until after you've run the IP. You definitely want as little proteolysis as possible before and during the IP.


Ok. Our IP protocol includes proteinase K after binding of the antibody with the 5methyl-cytosines.




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