Is it possible to shear DNA by french press to get fragment size about 500 bp?
I really doubt it, physically shearing DNA below a certain size is actually fairly difficult to do and I know large molecular weight DNA makes crude cell lysates from a french press pretty viscous. I believe you'd have to go with some pretty intense sonication.
I agree with Alexandra. A few years ago I used to break lot of E. coli with french press, with pressure up to 20,000 PSI. I had to regularly add DNAse to reduce viscosity, suggesting genomic DNA was released. You could use sonication or restriction enzyme.
Yoram
Thank you for your answers. I need fragment DNA about 500 bp and at the same time preserve proteins. When I used 100% amplitude, I got DNA about 500 bp, but proteins were demaged. Now I am trying 40% amplitude and DNA is about 800 bp. Do you have some experience with another amplitude?
And do you think that shearing by french press will have influence on subsequently sonication?
I did not try shearing DNA by sonication but I did do a lot of protein work after such. I would guess the problem is not amplitude but heat generated through sonication (you are pushing energy into the system...). So I suggest putting the tube with your sample in ice-water mixture (the water have much better thermal conductivity then the air between the ice). I usually use a 1.5 ml Eppendorf tube placed inside a float to prevent water into the sample. Now use 100% but do cycles of 10 seconds sonication + 50 seconds rest for chilling, 4-5 of those.
As of the sonication after french press, I do not think DNA will be effected, proteins might be effected from the fast pressure drop, depending what pressure you use. Personally I find french press unpractical for low cell concentration/amount.
Yoram
Also, when you sonicicate your samples you should use protease inhibitors like Sigma's inhibitor cocktail (P8465). You may also want to add at least 1 mM PMSF to be on the safe side.
Thank you Yoram. The problem Pavla may have is if she needs to use her protein(s) for a specific purpose, like in an animal model or a pharma product. Since protease inhibitors are highly toxic she may want to use something you can get rid of like high molar urea. In that case she may need to test if the protein(s) can be refolded after dialyzing or salting out the extract.
Thank you for comments. I am doing ChIP assay. Of course I am using protease inhhibitor. I need DNA ybout 500 bp, preserve protein function and preserve bound between my protein and DNA. It is difficult to determine optimal conditions.
I read once a work where they used glutaraldehyde to cross-link DNA and protein and study the interaction. If of interest I will try to find it.
Yoram
I came across this article by Aric Joneja and Xiaohua Huang which I think provides some relevant info.
BioTechniques, Vol. 46, No. 7, June 2009, pp. 553–556
https://www.biotechniques.com/multimedia/archive/00050/BTN_A_000113123_O_50754a.pdf
Thank you, I will be thankful for each advice. So far I use 1% formaldehyde for crosslink protein-DNA.
Also thank you, Sameer, for article about shearing DNA.
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