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Bacteria Lysis Reagent For RNA extraction - Need Expert's Opinion (Dec/01/2009 )

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Hi all, I'm new in RNA extraction.

I am trying to extract bacteria RNA. However, protocols I had read so far requires TE buffer and lysozyme.
I was wondering if there is any other recipe for Bacteria lysis prior to RNA extraction (without involve lysozyme).

Also, alkaline lysis (NaOH +SDS) was used in plasmid extraction but I have yet to see it was being use in RNA extraction...is there any explanation for this? Thank you.

Awaits your suggestion.

Adrian
Attached Image

-adrian kohsf-

Hi Adrian

Alternatively you can try bead beating to break the cells and proceed the extraction..But in my experience, lysozyme is the better way to do it. What bacteria do u work on?

-gogreen-

Hi gogreen,
I'm dealing with B.pseudomallei. Besides Beads beating, is there any chemicals which I can try on?

There is DNA lysis buffer recipe contain: "Triton-X 100, EDTA, Tris-cl"... will it work for RNA isolation?

Source: http://www.cbs.umn.edu/labs/perry/Protocols/bactPCR.html
Attached File

-adrian kohsf-

Hi Adrian, Just googled to find that its a gram negative bacteria...so it might be easier to break the cells, you can give a try with the lysis buffer, try to do that at 65 degrees for 10 minutes with constant vortexing..My experience with bacterial RNA isolation methods is that most of them work, but the extent to which it works differs..The key is to lyse most of the cells in the suspension to get maximum yield. The lysis might not be very efficient in first place, you might have to do a lot a trials. Can I ask why you dont choose lysozyme or beads which are tried and tested methods and why u wanna try a new protocol?

-gogreen-

Adrian,
Have you tried any commercially available kit and fond it didn't work?

-molgen-

Hi gogreen & molgen,

The reason I try not to use lysozyme is because I have to prepare it fresh. I try to avoid the beads vortexing to avoid generate aerosol as my bacteria is quite dangerous and pathogenic. After the cell lyses I try to use Trizol method with column purification. I afraid that certain chemicals will degrade RNA and affect downstream process such as Real-time PCR.

My experience with Promega SV Total RNA Isolation System and GeneMATRIX Universal RNA purification Kit (Eurx) gives genomic contamination. Maybe I had load too much of cells in it, even after DNAse treatment (Promega) I still can't get rid of it. This is the reason I was try to use a trizol + column method as was suggested in the forum: http://www.protocol-online.org/forums/inde...c=8032&st=0

Any comments or suggestions?

-adrian kohsf-

hi Adrian,

I used to store Lysozyme for over a month after dissolving it in Tris...But you can also buy lysozyme in solution which can be stored quite easily. I would choose lysozyme over anything else for the ease of use and it works for any bacteria..
for the gDNA carryover, I think if you take only an aliquot of the RNA after quantification, say 5 ug for the DNase treatment, you can get much cleaner RNA than taking the whole thing (I was assuming that you process the whole thing)

-gogreen-

gogreen on Dec 2 2009, 02:50 PM said:

hi Adrian,

I used to store Lysozyme for over a month after dissolving it in Tris...But you can also buy lysozyme in solution which can be stored quite easily. I would choose lysozyme over anything else for the ease of use and it works for any bacteria..
for the gDNA carryover, I think if you take only an aliquot of the RNA after quantification, say 5 ug for the DNase treatment, you can get much cleaner RNA than taking the whole thing (I was assuming that you process the whole thing)


Hi gogreen,
:) :) ;) :P :) :D Thanks for your idea of using only aliquot for DNase treatment...you are right, I process the whole thing...

You mention the lysozyme in solution which can be purchase and store. can you share with me the brand or product?

Thanks again.

Adrian

-adrian kohsf-

hi Adrian


Good to hear it was of some help! ;)
I remember seeing a lysozyme solution from sigma...but could not track that in their websit now...But here is one from Epicentre .. http://www.epibio.com/item.asp?ID=338

-gogreen-

Hi gogreen,

Thanks for your help. I glad to know a RNA expert like you.

Just one more thing...how you make your lysozyme buffer? got reference or protocol to share?

Thank you.

Adrian

-adrian kohsf-
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