Bacterial lysis for DNA extraction - (May/14/2001 )
I am having a problem in the lysis of a moderatelly halophilic strain that I am using to prepare DNA. It is Gram negative strain but it resists all kind of things that I tried, any help will be great.
I don't know about your cells, but there are quite a number of method for cell lysis - alkaline lysis, triton lysis, phenol treatment, boiling method as well as commercial kits, although these are primarily for coli and yeast. There are also mechanical methods such as sonication and french press. Most of these methods you can find on the web.
Resuspend bacteria in Tris pH 8 50 mM. Freeze and thaw (3-4 times) in 37°C bath and dry ice-ethanol. Then add proteinase K (at least 50 ug/ml) , incubate for 30 min at 37 or 50 °C. then perform fenol extraction. precipitate DNA with ethanol. collect DNA rapidly on a glass rod by vigorous stirring.Good luck.