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Cell Lysis for DNA extraction


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5 replies to this topic

#1 Julio-Claudian

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Posted 27 January 2010 - 12:06 AM

Dear all,

I've been processing my own batch of bacteria (Gram negative) without much problem and the DNA extraction were smooth.

Now, being given a batch of a few Gram positive bacteria, it's been frustrating getting them to break up. Of course, the Promega kit which I'm using suggested the addition of lysozyme. Problem now is what kind of lysozyme to use?

This link (Sigma) shows a great deal of lysozyme: http://www.sigmaaldr...p...;N25=0&F=PR

So, to those with experience breaking up the Gram positive bacteria, do help me out. Please? Other suggestions of lysis are welcomed.

Thanks people!

:D

#2 gebirgsziege

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Posted 27 January 2010 - 12:59 AM

not having much experience with bacteria, but I just used the cheapest lysozyme as we had some of it in our lab alreay (Order # 62971) and it worked well with my bacteria (although another group at my dep. was not successful with this lysozyme, they are using a special one from promega or quiagen). But I was doing 16S sequencing only, so probably the kind of lysozyme might be crucial for other applications.....
A man cannot be too careful in the choice of his enemies. (Oscar Wilde)

#3 mdfenko

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Posted 27 January 2010 - 07:23 AM

personally, i prefer the highest purity (but i don't work with gram positive bacteria).
talent does what it can
genius does what it must
i do what i get paid to do

#4 microgirl

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Posted 27 January 2010 - 11:20 AM

Try some freeze/thaw cycles too.

#5 Julio-Claudian

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Posted 29 January 2010 - 09:37 PM

Very well, thanks for the replies..

#6 gogreen

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Posted 01 February 2010 - 04:48 AM

I used to do a lysis with lysozyme conc of 3-4 mg/ml for about 20 min for my RNA isolations and it works for most of the of grm +ve bacteria




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