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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

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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.



#2 Trof

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Posted Yesterday, 03:14 PM

What kind of bacteria? Is it Gram-positive?

AFAIK you don't need homogenization for G- cells at all, and Trizol protocol recommends not to wash them prior with anything, just centrifuge out of medium. Though depending on the nuclease content and other things you may need to optimize that.

 

Also, why do you plan to centrifuge Trizol? Are there some debris you know Trizol won't lyse? In my experience Trizol eats almost anything, but I don't lyse bacteria in it.

 

There is also this product, that is designed to work with Trizol to lyse all kinds of bacteria, as I found now.

https://www.lifetech...roduct/16122012


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