3 replies to this topic

[AussieUSA]

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

[MKR]

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 .

AussieUSA, on Apr 16 2009, 11:11 AM, said:

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

[KPDE]

AussieUSA, on Apr 16 2009, 11:11 AM, said:

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

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.

[MKR]

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

KPDE, on Apr 16 2009, 04:35 PM, said:

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.