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DNase activity


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#1 kajmak

kajmak

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Posted 06 May 2011 - 01:37 AM

I canít get my head around DNase activity assay.

The group did crude cell extraction using 10 mM Tris-HCL, 3 mM MgCl2 and 2 mM 2-mercaptoethanol and centrifugation at 10 000 g for 10 min. Supernatant was incubated with some calf thymus DNA and 100 mM sodium phosphate buffer for 2 h at 37 C.

The reaction was stoped with perchloric acid (final concentration 4%) and concentration of acid soluble DNA measured at 260 nm.

What I donít get is, what is the point of 2 hour incubation if DNA is soluble in that acid? Isnít that extraction a little bit to crude and wouldnít 2-betacaptoethanol in such environment be able to facilitate DNase inhibition?

Thank you




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