Cells are lysed by sonication and further I want to remove DNA from lysates? What is the best method without damaging/reducing protein/enzyme activities?
I always add DNAse to my cells prior to lysis, regardless of whether I use sonication, a cell disrupter, a french press or BugBuster. If for some reason you also need to remove RNA you can either add RNAse A on top of the DNAse 1 or just go for Benzonase, which has both DNAse and RNAse activity and works really well (though it is a bit pricey).
Though I have heard that sonication should reduce most of the large DNA strands, I find that without DNAse it still produces somewhat viscous samples (it's the DNA released from the lysed cells that makes the cell lysate viscous). The viscosity makes it more difficult to separate the soluble fraction from the insoluble fraction using centrifugation and also clogs up any filters you might need to use before loading the soluble fraction onto a purification column.
If you used sonication to lyse your cells the DNA will be fragmented anyway. Unless your enzyme activities are going to be affected by small oligos, I would not worry about it. You cant make good DNA from sonicates.
I always add DNAse to my cells prior to lysis, regardless of whether I use sonication, a cell disrupter, a french press or BugBuster. If for some reason you also need to remove RNA you can either add RNAse A on top of the DNAse 1 or just go for Benzonase, which has both DNAse and RNAse activity and works really well (though it is a bit pricey).
Though I have heard that sonication should reduce most of the large DNA strands, I find that without DNAse it still produces somewhat viscous samples (it's the DNA released from the lysed cells that makes the cell lysate viscous). The viscosity makes it more difficult to separate the soluble fraction from the insoluble fraction using centrifugation and also clogs up any filters you might need to use before loading the soluble fraction onto a purification column.
HI Ravinder!
All answers are valuable, however I stringly agree with Paul and Antonio, specially using Benzonase.
Good luck,
You can use streptomicine sulfate precipitation. 2% for 30 min with gentle shaking.
I suggest PEI preicpitation. Brief protocol is given here.
0.1% polyethyleneamine (PEI) precipitation for half an hour while stirring it continuously on ice followed by centrifugation at ~12,000 rpm for 30 minutes at 4oC.
For no-hassle DNA removal look here: http://www.protean.cz/en/recombinant-protein/11/endonuclease-s-marcescens
You may precipitate with streptomycin sulfate (1%w/v) 30 min, 4º, with gentle shaker.
But watch out if your protein are a DNA binding protein. In htis case use 0,2%.
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