Sauer:Lysing E. coli with Lysozymes: Difference between revisions

Sauer:Lysing E. coli with Lysozymes (view source)

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 In most cases, simply adding lysozyme to your resuspended cells and waiting is sufficient to invoke lysis.  Some protocols call for placing the cells on ice during lysis in an effort to curb proteolytic activity.  This never made much sense to me,  you also slow down the activity of the lysozyme.  I have never seen a comparison of the activities of proteases and lysozyme at 0 and 25 degrees, but I generally lyse at room temperature.
-Lysis is apparent by a reduction in turbidity and a severe increase in viscosity from the liberated chromosomes.  For protein preparations, I usually add Benzonase because it has activity toward double- and single-stranded DNA and RNA.  If you just add DNase, be aware that most are purified from pancreas and are loaded with proteases.  If you see a lot of damage to your protein during lysis, add proteiease inhibitiors and/or change nuclease.
+Lysis is apparent by a reduction in turbidity and a severe increase in viscosity from the liberated chromosomes.  For protein preparations, I usually add the nuclease Benzonase to reduce the viscosity because it has activity toward double- and single-stranded DNA and RNA.  If you just add DNase, be aware that most are purified from pancreas and are loaded with proteases.  If you see a lot of damage to your protein during lysis, add protease inhibitiors and/or change nuclease.
 ===Intracellular Lysozymes===