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Old 11-27-2009, 12:33 AM   #4
aleidenroth
Junior Member
 
Location: Nottingham, UK

Join Date: Nov 2009
Posts: 8
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Thanks both.

@ECO
We have tried the <1.5kb conditions but got pretty big fragments.
I'll try and drop the intensity though. To get the time down, would you drop cycle time or cycle number?

@hamid
1) 1.5ml standard eppendorfs
2) 4x 10^6 cells, although I might increase that. Spending a lot of time waiting for my cells to grow at the moment...
3) I first lyse cells (5mM PIPES, pH8, 85mM KCl, 0.5% NP-40) and then nuclei (50mM Tris-Cl pH8, 10mM EDTA, 0.8% SDS)
4) I shear cells in the nuclear lysis buffer from 3)
5) Do you mean after the sonication? I haven't done any IPs yet, still at a very early stage.
6) We tested different cycle numbers (5, 10, 15, 20, 25, 30), and actually they all look pretty similar. We get slightly lower smears with higher cycle numbers.

Thanks for the linked protocol, I had seen that but used the other one (for SDS-buffers), which now seems to have disappeared off the website.

I'll try those conditions and see how low I can drop the cycle number.

Thanks for the quick feedback,

Andy
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