How to get rid of DNA contamination in protein samples - (Aug/28/2006 )
Hi,
I'm purifying a recombinant His-tagged (DNA-binding) protein for NMR studies and I'm having problems with DNA contamination after the affinity purification.
After lysis by sonication I want to incubate with DNase I (grade II) on ice.
Does anyone know how much (how many units of) DNase to use per wet weight of E. coli cells?
Hi,
I am not sure if this helps, but I have used Benzonase for digestion of DNA and RNA in protein samples. 50 U per 100 µl cell lysate and 1-2 mM Mg2+(but depends on you kind of benzonase). I also used RNAse H and DNAse I for digestion, ca. 10U per 100 µl of cell lysate. Can't say anything to the wet weight, perhaps you have to try with different amounts of enzyme.
I am not sure if this helps, but I have used Benzonase for digestion of DNA and RNA in protein samples. 50 U per 100 µl cell lysate and 1-2 mM Mg2+(but depends on you kind of benzonase). I also used RNAse H and DNAse I for digestion, ca. 10U per 100 µl of cell lysate. Can't say anything to the wet weight, perhaps you have to try with different amounts of enzyme.
Thanks! I'll Give it a try.
"Shear DNA by passing the lysate through a 18-gauge needle four
times. (Optional) If the lysate is very viscous, add RNase A (10 µg/ml)
and DNase I (5 µg/ml) and incubate on ice for 10-15 min instead."
From a protocol for purification of a tagged protein