The viscosity of the DNA can be reduced by shearing it. This can be done by pipetting the solution, or by a second passage through the French Press.

Thanks for your comment, Richard! Usually we pass the cells two to three times through the French Press. Just to ensure that most of the cells has been disrupted. However, always with DNase as mentioned. So will proceed just like always but without DNase.

Hi; cell lysis happened fine without using DNase1 when I did mine using JM109/DE3 cells; Composition of the lysis buffer was: 50 mM Tris, pH 8.0; 5 mM EDTA,0.25 mM PMSF,1 mM DTT, 20micrograms/ml lysozyme, and Triton x-100 to0.1%. Lysis was left 1 hr on ice before taken to french press.

Thank you guys for your valuable comments and inputs!

Meanwhile we have done lysed the cells by FrenchPress and it worked just like a charm. No DNaseI needed, as you all mentioned. Thanks again.

To David and Aruna:

I might have added to the confusion by writing "E. coli S12 extract". S12 is not referring to the strain designation. But to the method of preparing the cell-free extract (in short, the disrupted cells are centrifuged at 12.000 xg; see also Kim et al, 2006 [http://www.sciencedirect.com/science/article/pii/S016816560600469X]). We used an E. coli BL21 (DE3) strain, for the reasons David pointed out.

hi Janosch,

I am curious to know the results from cell free experiment you attempted..need your inputs in preparing cell extract..