Sonication for FastChIP - (Mar/04/2008 )
I am new in the ChIP ,I tried normal ChIP before and now I want to follow the Fast ChIP protocol in Nature protocols (Thanks KPDE) .I used 1% formadehyde and fixed 10 mins in RT before and I had no much trouble on the sonication. But now I fixed all my cells with 1.4% formadehyde and 15 mins in RT . And I can not get good sonication now. Can anyone give me some suggestion on it?
Attachment is a pic of my sonication.I got a lot of small fragment (200bp) and large fragment( larger than 4kb),how can I get the smeal in the ranger of 200-500bp-1KB ?
Many thanks!
[attachment=4322:sonicati...hr_35min.jpg]
I used a Sonicator Misonix 3000 ,and I used power 4,total time is 15 seconds,0.8s pulse on,5 sec pulse off,2-5 rounds (2 minutes rest between two rounds)
I already fixed a lots of cells in 1.4% ,the cells are difficult to culture and I do not want to culture these cells again and to fix in 1.% again. I want to get a good condition with these cells fixed in 1.4% and 15min RT. All suggestions are welcomed.
Attachment is a pic of my sonication.I got a lot of small fragment (200bp) and large fragment( larger than 4kb),how can I get the smeal in the ranger of 200-500bp-1KB ?
Many thanks!
[attachment=4322:sonicati...hr_35min.jpg]
I used a Sonicator Misonix 3000 ,and I used power 4,total time is 15 seconds,0.8s pulse on,5 sec pulse off,2-5 rounds (2 minutes rest between two rounds)
I already fixed a lots of cells in 1.4% ,the cells are difficult to culture and I do not want to culture these cells again and to fix in 1.% again. I want to get a good condition with these cells fixed in 1.4% and 15min RT. All suggestions are welcomed.
You can still use your old crosslinking conditions (1% for 10min) if that works for you and the protein you are trying to ChIP. Several people who have used Fast ChIP use those conditions. If you do want to use our conditions though, I would suggest increasing the power output to 5 or even 6 and seeing if that higher molecular weight stuff goes away. The band you see at 200bp or less is probably RNA and should be ignored.
Attachment is a pic of my sonication.I got a lot of small fragment (200bp) and large fragment( larger than 4kb),how can I get the smear in the ranger of 200-500bp-1KB ?
Many thanks!
[attachment=4322:sonicati...hr_35min.jpg]
I used a Sonicator Misonix 3000 ,and I used power 4,total time is 15 seconds,0.8s pulse on,5 sec pulse off,2-5 rounds (2 minutes rest between two rounds)
I already fixed a lots of cells in 1.4% ,the cells are difficult to culture and I do not want to culture these cells again and to fix in 1.% again. I want to get a good condition with these cells fixed in 1.4% and 15min RT. All suggestions are welcomed.
You can still use your old crosslinking conditions (1% for 10min) if that works for you and the protein you are trying to ChIP. Several people who have used Fast ChIP use those conditions. If you do want to use our conditions though, I would suggest increasing the power output to 5 or even 6 and seeing if that higher molecular weight stuff goes away. The band you see at 200bp or less is probably RNA and should be ignored.
Thanks! I will follow your suggestion.
Hi Chubuyi,
could you pm me the article with the fast ChIP protocol, please?
Would really be great if I could fasten up the IP part....
Cheers
kylvalda
Attachment is a pic of my sonication.I got a lot of small fragment (200bp) and large fragment( larger than 4kb),how can I get the smeal in the ranger of 200-500bp-1KB ?
Many thanks!
[attachment=4322:sonicati...hr_35min.jpg]
I used a Sonicator Misonix 3000 ,and I used power 4,total time is 15 seconds,0.8s pulse on,5 sec pulse off,2-5 rounds (2 minutes rest between two rounds)
I already fixed a lots of cells in 1.4% ,the cells are difficult to culture and I do not want to culture these cells again and to fix in 1.% again. I want to get a good condition with these cells fixed in 1.4% and 15min RT. All suggestions are welcomed.