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Old 12-23-2012, 06:27 AM   #12
nfourier
Junior Member
 
Location: Israel

Join Date: Dec 2012
Posts: 8
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Hello Hamid,
1) I fix my cells in their medium, in formaldehyde (1.42% final concentration) for 15min in room temperature. It had worker before with 3 different cell lines in our lab.
2) I prepare the nuclei with my IP buffer (according to the FastChIP protocol): 150mM BaCl, 50mM Tris-HCl (pH 7.5), 5mM EDTAm NP-40 (0.5%), Tirton X-100 (1%). I resuspend 10^7 cells pellet in 1ml buffer and centrifuge (12K G, 1min). Resuspend again, centrifuge, and then use the same buffer as shearing buffer.
The last time I did the protocol I used the shearing buffer you suggested.
Again, this protocol has worked for us many times before.
3) For DNA analysis I do ethanol percipitation and use Chellex (as in the FastChIP protocol). Because I don't do RNase treatment I do see the RNA on my gel around the 100bp size.

I am aware of the kit, but since our protocol has worked before I am reluctant to change to a new kit.
I am familliar with Ethan's protocol. His lysis buffer is very similar to mine (a little different % in Triton X and NaCl) and his shearing buffer is like the one you recommended. He used 5% duty cycle and 12min on Covaris S2, while I did it for 8 min. While he uses TC12X12 tube I used the microTubes. Maybe that's the problem?
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