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Hi all,
Would welcome opinions on the following. I am trying to identify viable bacteria in solution after antimicrobials treatment to distinguish what has been killed and what has not. Dead bacteria will have DNA in solution, live bacteria will have DNA encapsulated in a cell. Wondering how I can seperate live cells from dead bacteria. Will perform identification using 16s sequencing. Ideas so far, ampure beads to bind free DNA, centrifugation of intact cells, circulating DNA kit for isolation, PMA/EMA methods, cell sorting, column purification of DNA without lysis buffer. Cannot use rt of rRNA as it seems to be stable in solution much longer than mRNA. Maybe an RNAse/DNAse treatment to remove external nuclei acids follow by heat inactivation and lysis?
Any suggestions as to these ideas, or any other brainwaves?
Would welcome opinions on the following. I am trying to identify viable bacteria in solution after antimicrobials treatment to distinguish what has been killed and what has not. Dead bacteria will have DNA in solution, live bacteria will have DNA encapsulated in a cell. Wondering how I can seperate live cells from dead bacteria. Will perform identification using 16s sequencing. Ideas so far, ampure beads to bind free DNA, centrifugation of intact cells, circulating DNA kit for isolation, PMA/EMA methods, cell sorting, column purification of DNA without lysis buffer. Cannot use rt of rRNA as it seems to be stable in solution much longer than mRNA. Maybe an RNAse/DNAse treatment to remove external nuclei acids follow by heat inactivation and lysis?
Any suggestions as to these ideas, or any other brainwaves?