ChIp on mouse liver - (Mar/01/2008 )

Hello
I am new on this forum and I am looking for someone who could help me to perform chip on fresh mouse liver.
I tried during several month to cross link after collagenase perfusion but it didn't work.
Now I am performing perfusion with formol but I don't know how many grams to resuspend in lysis buffer. The whole liver seems too much.
Is there someone who "chips" successfully on fresh mouse liver who could help me?
Thanks

-ChipLiver-

QUOTE (ChipLiver @ Mar 1 2008, 08:43 AM)

Here is a thread to look at where your question is likely addressed:

http://www.protocol-online.org/forums/inde...showtopic=20402

This is what I posted in that thread regarding how a colleague in my lab processes rat livers for ChIP:

QUOTE (KPDE @ Sep 27 2006, 11:27 AM)

A researcher in my lab takes the frozen tissue fragment, immerses it in 1% Fromaldehyde in PBS and quickly minces it into small pieces with scizzors. He crosslinks for 20 min, quickly pellets the tissue fragments and aspirates the supe, and then quenches with glycine in PBS. He's gotten consistent results with this method of crosslinking. As you say, the issue is equal access of the formaldehyde to the different layers of the tissue so the mincing is very important.

-KPDE-

thank you for your help but I prefer to perform crosslink on fresh liver and then isolate hepatocytes.
Does anyone have an idea of how many grams I should use per ml of lysis buffer an by IP?

-ChipLiver-

QUOTE (ChipLiver @ Mar 3 2008, 02:24 AM)

thank you for your help but I prefer to perform crosslink on fresh liver and then isolate hepatocytes.
Does anyone have an idea of how many grams I should use per ml of lysis buffer an by IP?

Hi,

Sorry I didn't read your post more carefully. The amount of tissue my colleague uses is approx. 200-300mg in 1ml buffer and from this he uses 60-80ul per ChIP. If you don't get any help from anyone who uses fresh liver, this might be helpful.

-KPDE-

thanks
I'm gonna try and I will tell you.
(I tried yesterday but DNA is not sheared enough).
I'm using a bioruptor and I'm surprised to see that people don't sonicate more than 10 minutes whereas I need at least 15minutes.
my chromatin is maybe overfixed

-ChipLiver-

QUOTE (ChipLiver @ Mar 5 2008, 01:45 PM)

my chromatin is maybe overfixed

How long do you fix the tissue for and at what temperature?

-KPDE-

I perfuse the liver during 5 minutes ar RT and then I cut the liver in small pieces and then incubate 10 minutes at RT

-ChipLiver-

QUOTE (ChipLiver @ Mar 6 2008, 08:42 AM)

I perfuse the liver during 5 minutes ar RT and then I cut the liver in small pieces and then incubate 10 minutes at RT

Hmmm. That doesn't seem too long at all. What concentration of formol are you fixing in? Is it the typical 1 to 1.5% formaldehyde?

If you're not over fixing could it be that you aren't sufficiently homogenizing the tissue prior to sonication? One of the people in my lab uses a Tissue Tearor before sonication and this works well (kidney and some liver samples).

-KPDE-

Formaldehyde 1%
I use a glass dounce to homogenize the tissue and it s sure that the tissue is not perfectly homogenized.

-ChipLiver-

I have another question, I would like to know which CT do you usually obtain for input DNA?
I use 5ul of 1/10 and 1/100 input DNA, and I get get 28 cycle for a lot of primers. More DNA does'nt allow to obtain earlier ct.

-ChipLiver-