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Animal tissue ChIP - to freeze or not to freeze?


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#1 AussieUSA

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Posted 16 April 2009 - 10:11 AM

I am newish to ChIP but an old hat to IP and PCR etc. I am going to look at a transgenic mouse that seems to have developed a compensatory mechanism for the gene that has been modified thus I will use ChIP to see which transcription factor is now binding to my gene(s) of interest.

My question is: Can I snap-fresh my tissue during the tissue harvesting procedure, transfer to -80˚C freezer and process (fix and lyse) a day or so later :( ?

This would make life easier as I am collecting tissue at different time points :) .

Thanks,

AussieUSA

#2 MKR

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Posted 16 April 2009 - 12:24 PM

Interesting that you ask this question, because I always have used dry ice to quickly freeze brain tissue and then store it in -80. My ChIP has worked, but at first (when it wasn't) I was very worried about whether I should do the fresh tissue thing. I think, at least for me, it works fine to freeze first. But even now, sometimes I wonder. I also wonder about fresh versus frozen tissue for nuclear extraction using a hyptonic solution with NP40. But I always do end up using frozen :).

I am newish to ChIP but an old hat to IP and PCR etc. I am going to look at a transgenic mouse that seems to have developed a compensatory mechanism for the gene that has been modified thus I will use ChIP to see which transcription factor is now binding to my gene(s) of interest.

My question is: Can I snap-fresh my tissue during the tissue harvesting procedure, transfer to -80˚C freezer and process (fix and lyse) a day or so later :( ?

This would make life easier as I am collecting tissue at different time points :) .

Thanks,

AussieUSA



#3 KPDE

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Posted 16 April 2009 - 03:35 PM

I am newish to ChIP but an old hat to IP and PCR etc. I am going to look at a transgenic mouse that seems to have developed a compensatory mechanism for the gene that has been modified thus I will use ChIP to see which transcription factor is now binding to my gene(s) of interest.

My question is: Can I snap-fresh my tissue during the tissue harvesting procedure, transfer to -80˚C freezer and process (fix and lyse) a day or so later :lol: ?

This would make life easier as I am collecting tissue at different time points :o .

Thanks,

AussieUSA


I process frozen tissue for ChIP and so far it has worked great. For liver tissue I quickly mince the tissue with forceps in 1% formalehyde and let it incubate at RT for 20 minutes. I spin and remove the form and add 125mM glycine and incubate for 5 min. Then spin and wash twice with PBS. After that it's ready to sonicate.

#4 MKR

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Posted 16 April 2009 - 09:13 PM

Wow, that's almost exactly what I do! I have frozen brain tissue to which I add 1% formaldehyde in PBS, 20min, spin/remove, add glycine in PBS (twice to wash and quench cross linking), but then I use RIPA and sonicate :lol:


I process frozen tissue for ChIP and so far it has worked great. For liver tissue I quickly mince the tissue with forceps in 1% formalehyde and let it incubate at RT for 20 minutes. I spin and remove the form and add 125mM glycine and incubate for 5 min. Then spin and wash twice with PBS. After that it's ready to sonicate.






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