DNAse I incubation - shortest sufficient time - (Jul/01/2008 )

we have to shorten the incubation time for DNA cleavage by DNAse I in cell lysates since we fear proteolyses;

which protocol for DNase I you are using (units? time? temperature?)?

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I doubt you will have complete DNA digest in the crude cell lysate due to accessibility. But this question may be answered by experimenting. What lysis buffer are you using and will it interfere DNase I digest? Why do you need to digest the DNA in the first place? The lysate too sticky to precess?

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yes, to sticky total lysate; we now need more protein per lane than usual but sample after boiling was too sticky even if you pipet it hot; I think it is cytoskelton proteins but also DNA; we use a normal Tris-buffered 0.1% triton x-100 lysis buffer with protase and phosphatase inhibitor cocktails from Roche, and 0.5% DTT

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I am not certain whether your lysis buffer contains Ca2+ and Mg2+; so here goes.. When I treat my samples (crude nucleic acid preps) with DNase I I use this buffer (composition from Ambion). Ca2+ ions are vital for DNaseI activity and Ambion says "evidence in the literature suggests that what little DNase I activity is present in buffers lacking Ca2+ is due to synergistic activation by contaminating Ca2". I have found better DNase activity in this buffer; so perhaps using this buffer could help you reduce your incubation time.

10X DNase I Buffer
100 mM Tris pH 7.5
25 mM MgCl2
5 mM CaCl2

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Bear,

If you use AquaRNA to extract proteins, you won't have any of those problems. It's an extremely potent protein denaturant and you don't need any protenase inhibitors. After cell lysis, simply add 1 vol of isopropanol to remove DNA/RNA. The proteins remain in the supernatant and they are then recovered by acetone precipitation. Here's the protocol.

Good luck!

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thanks for protocols but the protocol for protein preparation will definitely lose some cytoskeleton proteins (discriminated as insoluble fraction) but we need them, too

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thanks for answer; lysis buffer try to reduce Ca2+ and Mg2+ to protect against metalloproteinases but EGTA/EDTA do only buffer so that there will be a some free Ca2+ and Mg2+ left from cells and rests of medium; this may be actually enough for (suboptimal) working of DNAse I

in the mean time we have worked with an excess of DNA I, and succeeded in reducing some viscocity

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The final insoluble formed in the protocol is not protein but the components of AquaRNA. Both soluble protein and structure protein are recovered, ex., tubulin is near 100% recovered from the lysate.

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so, not only DNA but RNA are making trouble; do you recommend the use of RNAse? are both RNAse and DNAse to apply in parallel?

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No, RNA is not the problem. They've gone as soon as you are done with the lysis using your lysis buffer. The gluey stuff is DNA. But because of the viscosity and protein binding, your DNase may not cut the DNA efficiently. That's why I suggested you give AquaRNA a shot, it literately peels off the proteins from the DNA.

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