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Can I go for ChP having frozen tissue - (May/18/2012 )

Hi Friends,
I m working on histones and wanted to do ChiP. I have frozen tissue samples can I use -80frozen samples in ChiP


Best Regards

Binsan

-pakbiochemist-

Possibly.

You might try crushing and grinding them in liquid nitrogen and then transferring the powder directly to 1% formaldehyde in PBS. Crosslinking time varies depending on how fine you grind the sample (10-20min at RT).

Alternatively you can try thawing briefly in formaldehyde/PBS and mincing quickly (I suggest spring scissors if you're doing this in a microfuge tube). Because you can't get very fine pieces this way I suggest using the longer crosslinking times (15-20min).

-KPDE-

yes you can use them untill one year.
I usually do it.


-----
Babak Memari

-memari-

We do ChIP from frozen samples all the time. The tissue is allowed to thaw slightly on ice, minced on ice with sharp dissecting scissors, resuspended in DMEM (no FBS) then passed (scraped) through a nylon cell strainer into a 50 mL tube with ~10 mL cold DMEM, everything is kept on ice. Once the single cell suspension in DMEM is prepared formaldehyde is added to 1% final concentration and the protocol proceeds as normal.

-biznatch-

biznatch on Tue Jun 5 23:40:23 2012 said:


We do ChIP from frozen samples all the time. The tissue is allowed to thaw slightly on ice, minced on ice with sharp dissecting scissors, resuspended in DMEM (no FBS) then passed (scraped) through a nylon cell strainer into a 50 mL tube with ~10 mL cold DMEM, everything is kept on ice. Once the single cell suspension in DMEM is prepared formaldehyde is added to 1% final concentration and the protocol proceeds as normal.


Hi,
Can I ask why the tissue have to put in DMEM but not cold PBS or saline? And any difference between fresh tissue and snap frozen tissue? I will do NChIP for the both fresh and snap frozen tissue, any recommendation for me?

Thanks!

-kblee-