Loosing Cells with centrifugation? - (Mar/15/2006 )
I try to extract bacterial DNA from leaves. The protocol i use starts with mechanical disruption by using 0,7mm zirkonium bits. Then ther is a centrifugation fpr 6 minutes with 12'000 rpm, the supernatant is taken and than there is a treatment with lysozym.
Question: In my view the lysozym treatment after the centrifugation does not make sense, because the cells i want to break up with lysozym are in my pellet - or are there still enough cells in my supernatant?
I'm glad about your ideas!
I should think many of your bacteria will be in the pellet if you centrifuge at that speed/time
where did you find this protocol?
I don't know this protocol but it would make much more sense to me to add the lysozyme before the spin, possibly even before the mechanical disruption. I agree with you, I can't see any sense in adding the lysozyme to the super. Could it be possible that the pellet is retained and treated with lysozyme and the supernatant is discarded?
before the mechanical disruption - i dont think its so good, because than i enhance the risk to destroy my DNA by turbulence forces - i think the mechanical step is for getting the bacterial cells out auf the intercellualrs.
I dont think so, becaus I have the DNA in my supernatant, that I won with mechanical disruption (bits) and chemical treament (SDS) - it would be useful when i only want to get grampositives.
The protocol is a standard-protocol used at the University Giessen (germany) - i don't have a publication where it is mentioned,...