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Weird Problem with Fast ChIP


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#1 biorat

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Posted 06 January 2011 - 10:47 AM

Hi Everyone,

I've been trying fast ChIP protocol recently and I love it! The only problem I have is that my gel electrophoresis for evaluation of DNA shearing has been showing almost no signal. I have tried to use about 2*105 and 4*105 cell's chromatin as input and could not see any signal on gel. I once tried to use 2*106 cell's chromatin and could see a very faint smear. Despite this problem, PCR has been running pretty well with both input and pull-down sample.
Does anybody know what could be the problem of getting no bands/smear with fast ChIP?

Thank you very much.

RC

#2 angelawu

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Posted 06 January 2011 - 01:59 PM

Hi Everyone,

I've been trying fast ChIP protocol recently and I love it! The only problem I have is that my gel electrophoresis for evaluation of DNA shearing has been showing almost no signal. I have tried to use about 2*105 and 4*105 cell's chromatin as input and could not see any signal on gel. I once tried to use 2*106 cell's chromatin and could see a very faint smear. Despite this problem, PCR has been running pretty well with both input and pull-down sample.
Does anybody know what could be the problem of getting no bands/smear with fast ChIP?

Thank you very much.

RC


Are you using the Chelex extraction method, and then running the gel? If you are, then your DNA might be running off the gel, since the resulting DNA from Chelex extraction is single stranded (cuz it's been boiled). Try running it with a ssDNA ladder or RNA ladder and for less time if you want to size it. I use the RNA chip on bioanalyzer for my Chelex treated samples.

#3 biorat

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Posted 06 January 2011 - 02:30 PM

Thanks Angelawu.
Yes, I did chelex extraction and then run agarose gel. My first dsDNA marker (100bp)is only half way through the gel so I did not think the ssDNAs all ran off the gel. But I will try to run a gel with shorter time to see if this is the problem.

#4 angelawu

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Posted 06 January 2011 - 03:19 PM

Thanks Angelawu.
Yes, I did chelex extraction and then run agarose gel. My first dsDNA marker (100bp)is only half way through the gel so I did not think the ssDNAs all ran off the gel. But I will try to run a gel with shorter time to see if this is the problem.


Also, depending on the method you're using to visualize the DNA, it might not work on ssDNA. E.g. ethidium bromide is a DNA intercalator, so it will only work on dsDNA.

Edited by angelawu, 06 January 2011 - 03:20 PM.


#5 chabraha

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Posted 06 January 2011 - 04:00 PM

Try soaking your gel in an ethidium/TBE buffer for 30 min or so. I use the Chelex method and find that works fine using ethidum. Sometimes if your Chromatin is not sheared efficiently (ie-the DNA size ranges from 10kb-200bp) then the staining is so diffuse that you can barely see it
I use a non-chelex method to visualize my sheared chromatin and it takes ~2hrs..........its not as fast but its better than the O/N reverse crosslink method.....let me know if your interested
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#6 biorat

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Posted 07 January 2011 - 06:24 AM


Thanks Angelawu.
Yes, I did chelex extraction and then run agarose gel. My first dsDNA marker (100bp)is only half way through the gel so I did not think the ssDNAs all ran off the gel. But I will try to run a gel with shorter time to see if this is the problem.


Also, depending on the method you're using to visualize the DNA, it might not work on ssDNA. E.g. ethidium bromide is a DNA intercalator, so it will only work on dsDNA.


I am using ethidium bromide. That might be the reason why the signal is so faint. But I guess most of other people doing fast ChIP are also using ethidium bromide and having no problem. So I figure I am be doing something wrong.

#7 biorat

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Posted 07 January 2011 - 06:30 AM

Try soaking your gel in an ethidium/TBE buffer for 30 min or so. I use the Chelex method and find that works fine using ethidum. Sometimes if your Chromatin is not sheared efficiently (ie-the DNA size ranges from 10kb-200bp) then the staining is so diffuse that you can barely see it
I use a non-chelex method to visualize my sheared chromatin and it takes ~2hrs..........its not as fast but its better than the O/N reverse crosslink method.....let me know if your interested


Thank you chabraha.
I always mix ethidium bromide with agarose. Would soaking the gel in EB provide any benefit?
I tried to sonicate with the highest power setting for a total of 200 seconds (10sec * 20times) so I guess the chromatin might have been sheared enough (or was I wrong to make such assumption?)

2 hours is pretty fine for me. I'd appreciate very much if you could share with me the non-chelex method to visualize sheared chromatin.

#8 chabraha

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Posted 10 January 2011 - 10:33 AM

Biorat,

do you see DNA via EB in your un-sheared control? I have found that soaking my gel in EB/TBE helps significantly in visualizing my gels. I think i posted the 2hr fast protocol somewhere else in this forum, if you cant find it let me know and I will type it out again........200sec seems like much for tissue culture cells. I use 8-10 10sec pulses on a setting of 8 with a fisher sonic dismembrator.
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#9 nipa68

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Posted 20 March 2011 - 05:18 AM

Dear Chabraha,

I also want to try out the fast ChIP and I am happy that I coincidentally found this page and your hint for a chelex-free but only 2 hrs long method to purify the DNA to check for the sonication efficiency. Could you please post it again?

Thank you very much for your effort.

Nicole

#10 chabraha

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Posted 22 March 2011 - 06:33 AM

Nicole,

Spin sheared chromatin down at 14,000xg for 10min
Take 100ul of sheared sample & combine with 200ul 1.5X Szak's Crosslink Reversal Buffer
Incubate 1hr @ 100C
Let cool and add 20ug ProK, incubate at 57C for 1hr
Extract once with Phenol:Chloroform. Transfer 250ul to a new tube
Add 10ug glycogen, 1/10vol NaAcetate, and 2.5vol ice cold EtOH
Spin at max speed at 4C for 20min
Wash pellet with ice cold 70%EtOH
Resuspend pellet in 60ul Tris pH8
Load 40 and 20ul on a 1.2% agarose gel
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