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Re-sonication ?


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#1 Xenos

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Posted 15 May 2009 - 06:38 AM

Hi!

Maybe it's a dumb question but is it possible to re-sonicate aliquots of chromatin I've already sonicated and stored at -80C (prepared with EZ ChIP kit) ? My sonication wasn't optimal (smear too high on gel), and need 1 or 2 additionnal pulses. I guess repeated freezing/thawing will denature proteins but since I plan to ChIP it right after this second round of sonication I don't see why it should be a problem.
Any advice ?

Thanks a lot

#2 Clare

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Posted 15 May 2009 - 07:45 AM

Hello :)

We have not re-sonicated after freezing, but we have compared ChIPs on frozen vs fresh chromatin from the same patients and got the same results. Why don't you just give it a go and see what happens? I'd like to know :)

We always check our sonication efficiency before we start adding antibodies etc. So after we sonicate we take a small aliquot (equal to about 1 million cells) and do a 'quick reverse cross-linking' while the rest of our chromatin is pre-clearing. So NaCl/RNaseA for 1 hour and then PK for 1 hour and run it on a gel. If it needs sonicating again, we usually give it an extra minute or 2 (we use a Bioruptor), spin it down and then set up our IPs.

It's so worth doing this step if you're not sure if your sonication will work every time. ie: if you use a lot of different cell types etc.

Have a great weekend :)

Clare

Hi!

Maybe it's a dumb question but is it possible to re-sonicate aliquots of chromatin I've already sonicated and stored at -80C (prepared with EZ ChIP kit) ? My sonication wasn't optimal (smear too high on gel), and need 1 or 2 additionnal pulses. I guess repeated freezing/thawing will denature proteins but since I plan to ChIP it right after this second round of sonication I don't see why it should be a problem.
Any advice ?

Thanks a lot



#3 lsek

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Posted 31 May 2009 - 04:26 AM

Hi Clare,

What's the condition for "quick reverse cross-linking"? Do you phenol-chloroform extract after PK? Thanks in advance

:o

Edited by lsek, 31 May 2009 - 04:27 AM.

===><===


#4 Clare

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Posted 01 June 2009 - 12:35 AM

Hello :)

We never phenol-chloroform extract - we just purify using the QIAGEN PCR purification kit.
Clare


Hi Clare,

What's the condition for "quick reverse cross-linking"? Do you phenol-chloroform extract after PK? Thanks in advance

:)



#5 lsek

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Posted 01 June 2009 - 07:12 AM

Thanks Clare :)

===><===


#6 Clare

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Posted 01 June 2009 - 07:21 AM

Thanks Clare :)


No worries :)

#7 KPDE

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Posted 01 June 2009 - 09:46 AM

Hi Clare,

What's the condition for "quick reverse cross-linking"? Do you phenol-chloroform extract after PK? Thanks in advance

:)


For a very quick reversal of crosslinking (10 minutes) you can dilute the chromatin in a high pH buffer with a little chelating agent (25mM Tris pH10, 1mM EDTA) and heat at 100C for 10 minutes. This method is very reliable and works for both input and ChIP samples (for ChIP samples just add this buffer to the washed beads after IP and do your PK digestion followed by 100C for 10min). If you are using a large volume of chromatin for your inputs (higher than 10l to 100l of diluting buffer) you might want to use a 10% chelex-100 suspension instead of the Tris buffer.

Edited by KPDE, 01 June 2009 - 09:48 AM.





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