I need to quantify bacteria abundance in gastrointestinal biopsies based both in bacterial DNA and RNA. The problem is that for Gram+ sometimes is difficult to disrupt the cell wall and we get poor or negative results for some specific bacteria. Do you know any method to deal with this issue? Thank you
Hi
You can use "High pure template preparation kit" by Roche product number: 11796828001). Then extend the time of incubation with "Binding Buffer".
How ever, initially you need de-paraffinize the sample if you use Paraffin embedded tissue blocks.
I can Email you the modified protocol
To isolate DNA from gram +ve bacteria I suggest you incubate the sample in lysozyme sol. or a mixture of lysozyme + lysostaphin (btw 30m-1h @ 37 oC) before digesting in a proteinase sol. and then proceed with your regular standardised protocol.
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