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Bacillus subtilis DNA extraction protocol

bacillus subtilis

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#1 babyoctopus

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Posted 09 November 2011 - 04:15 AM

Hi, I'm doing Bacillus Subtilis and I'm required to extract the total DNA from it. Is there any efficient extraction method for Bacillus?

I've try phenol chloroform method and the ethanol precipitation. But the amount of DNA extracted is too little that no band is spotted after I run a gel. Need help.....

#2 Micro

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Posted 09 November 2011 - 04:52 PM

What lysis method do you use before the extraction? With Gram positive, spore formers this step can be important to getting a good quantity of DNA.

#3 babyoctopus

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Posted 09 November 2011 - 05:24 PM

I don't understand. I just grow it overnight and harvest the cell and just use the DNA extraction buffer and proceed with phenol chloroform and ethanol precipitation. Is there any lysis method required for bacillus? Because I'm given an e coli method to perform the extraction. But I get very very little amount. In the DNA extraction buffer contain Tris HCl, EDTA, SDS...

Or do I need to add in lysozyme?

#4 Micro

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Posted 09 November 2011 - 06:00 PM

In my experience E.coli is lot easier to extract without lysis of the cell wall than Gram positive (but I work with actinobacteria not Bacillus so every organisms has its little quirks).

Your on the right track with the the lysozyme. Try adding a lysis step with lysozyme it might take a few goes to get the method right for your organisms but looking at a method I use... wash your cells to remove broth from cell mass, resuspend cells in about 480uL of TE buffer (or something similar) and add 120uL of 20mg/mL lysozyme (suspendend in TE buffer and only made up just before you use it). incubate for 30-60mins at 37oC.... then go from there with your method.

You could try Googling bacillus and lysozyme and see if you can fing a better set of concentrations and times specific for what you work with.

Goodluck!

Edited by Micro, 09 November 2011 - 06:01 PM.


#5 babyoctopus

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Posted 09 November 2011 - 06:35 PM

Yes, previously I work with e coli and I got high concentration and purity but even I use an enhanced method of extraction e coli on Bacillus, it don't work out at all.

I decide to give a try with adding lysozyme into my extraction buffer and incubate it (Coz I google and they say lysozyme need to work together/work better with EDTA). I'm not sure whether what I'm doing the right thing, is it proper to add lysozyme in extraction buffer, incubate at 37 C and spin it down? And then continue with the chloroform extraction and ethanol precipitation.

Thanks a lot for the guidance!

#6 Micro

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Posted 09 November 2011 - 08:50 PM

Using the extraction buffer with the ingredients you describe in the first post shouldn't be a problem with lysozyme. SDS is a lysis agent in itself and there are methods out there that use the two together. You could also try proteinase K or if you have the resources available a mechanical disturbance method like bead beading.

When I've used lysozyme with phenol:chloroform extract I don't spin down the cells after the lysis step I just add the P:C at a 1:1 ratio with the cell lysate mix.

#7 Hira Muzzamal_27321

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Posted 11 December 2011 - 07:35 AM

Hy to all..!
i am also doing with bacillus n im getting the same problems of getting light DNA. i also had a suggestion
of lysozyme but this is not available to me.otherwise i had the same procedure of DNA extraction...
so kindly suggest any other solution to that to get a sharp band of high quality DNA so that i can do
PCR with that..
thanks...

#8 ChrisLancer_01

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Posted 21 December 2011 - 12:05 PM

Hi,

some people in my lab have been talking about this new kit called PureLyse from Claremont BioSolutions. The kit is for lysis and extraction. They boast in one of their data sheets that it can disrupt Bacillus subtilis in roughly 40 seconds with over 80% efficiency. It seems like it's mechanical lysis and solid phase extraction in two steps and you can go straight into PCR after the prep. Here's the link, I think they might still be giving out free samples of this. I requested a sample a few days ago. Let me know if this is of any use to you. http://www.claremont...reLyse_s/57.htm





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