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bacterial RNA isolation from PBS buffer water

Membrane filtration Membrane filter TRIzol Homogenization

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#1 Md Jibran Alam

Md Jibran Alam

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Posted 28 November 2014 - 05:38 AM

I want to extract the total  bacterial RNA directly from sample collected in PBS. I cant enrich the bacteria as my research focuses on direct isolation of rna. As bacterial count is less, i also need to filter out the bacteria.

 

I dont have any bead-beater in my lab. So, I cannot use MOBIO Powerwater kit to isolate rna from bacteria filtered with .22 um membrane filter.

 

I have TRIzol available in my lab. So I want to follow the steps below. Please tell me if it is okay or suggest modifications what I can do with TRIzol reagent.

 

Modified Homogenization steps by using TRIzol-

 

1. separate the bacteria with .22 um membrane filter (1 cm in diameter).

2. put the membrane filter in a microfuge tube.

3. add 750 ul TRIzol reagent inside the tube.

4. crash / homogenize the membrane filter with IQ plus tissue homogenizer.

5. centrifuge for 2 min at 13000 rpm. (???? need more duration??)

6. collect the supernatant from the tube and proceed to the subsequent manufacturer protocol of TRIzol reagent.

 

Thanks in advance.







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