Lookin for a genomic DNA extraction kit for bacteria

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Lookin for a genomic DNA extraction kit for bacteria

Postby genetim » Mar 05 2008 11:49 pm

Hi all,

I'm looking for a good genomic DNA extraction kit for bacteria. Do you guys have any suggestion?
By the way, what criterion do you have to choose a suitable kit?

Thx a lot.
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Postby Iwantcrts » Mar 06 2008 3:40 am

I generally use a home made phenol:Chloroform extraction protocol (Employing SDS and glass beads for cell lysis). This gives loads and loads of DNA, then I clean the DNA up with a commercial spin column. This has been used successfully for Archea (Sulfolobus), Bacteria (E. coli), Yeast (S. cerevisiae) and Cyanobacteria (too many to list).

In my experience commercial kits aren`t vigorase enough when it comes to cell lysis (esp for cyano DNA extraction, they have a thick peptidoglycan cell wall).
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Postby genetim » Mar 06 2008 8:51 pm

I see

This is a good idea

Thank you
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Postby Iwantcrts » Mar 07 2008 3:25 am

Another option would be to include your own lysis method at the start (if you try a commercial kit and find that yields are low). For this you could try Liquid N2 grinding or sonication.
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Postby genetim » Mar 07 2008 10:00 pm

Can these methods (N2 and sonication) be applied to all bacteria species?

Thank you for your suggestion
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Postby Iwantcrts » Mar 08 2008 6:12 am

Yes,

Liquid nitrogen grinding combines freeze-thaw lysis with the mechanical lysis imposed by grinding the frozen biomass.

Sonication will also work, using a probe that actually goes into the cell paste works best but a sonication water bath should also induce cell lysis (remember to put ice in it though to keep the temp down).
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Postby genetim » Mar 09 2008 9:09 pm

What a good method as I got liquid N2 and pestle and mortar in my Lab!

For me, time is an important parameter, both PCR and sequencing are required in downstream.

Thank you for suggestions.
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Postby 12260 » Mar 13 2008 1:56 pm

Try this:

-Make a 1ul suspension in 1ml PBS
-vortex
-Boil for 10 mins
-centrifuge at 14000xg for 5mins
-transfer supernatant into a fresh epindorf
-freeze at -20
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Postby genetim » Mar 18 2008 2:45 am

Can boiling break the cell wall and the cell membrane of the bacteria?

Thank you for your help
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Postby 12260 » Mar 18 2008 5:19 am

[quote="genetim"]Can boiling break the cell wall and the cell membrane of the bacteria?[/quote]

I imagine it probably does, but i think you need to get the correct timing. A 10 minute boil PCRed a dream for me. Use about a 1ul suspension, if your supernatant is yellowish then it probably contains proteins as you used too heavy an innoculation. Boiling for too long will probably degrade the DNA. 10 minutes should be fine for 1ml PBS.
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Postby genetim » Mar 19 2008 11:06 pm

I see

Thank you for your help, 12260
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Re: Lookin for a genomic DNA extraction kit for bacteria

Postby jcoalsonshore » Nov 08 2012 10:09 am

A company called cellcrusher produce a device for cell lysis in liquid nitrogen. It comes with a drill-bit accessory for motorised grinding. Good for yeast and fungus, probably over-kill for bacteria!

http://cell-lysis.com

http://cell-lysis.com/?page_id=65 (the demo video)
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Re: Lookin for a genomic DNA extraction kit for bacteria

Postby memari » Nov 15 2012 2:11 pm

This is a home made recipe:
Prepare this Lysis buffer (Chip Lysis Buffer):

50 mM Tris, pH around 8.0
10 mM EDTA
1% SDS


Then mix these:
50% of Phenol(pH around 7.8 )/Chloroform
25% of Lysis buffer (Chip Lysis Buffer)
25% of Water

Now you can use it for DNA extraction:
Add 1 ml of that mixture above with pellet of bacteria or cells.
Dissolve bacteria or cells by pipetting up and down.
Keep vials on ice for 10 min.
put vials on vortexer horizontally and vortex for 15 seconds.
Centrifuge >11000 g for 15 min
Take supernatant.
Add the same volume of Isopropanol and invert several times and stay for 10 min at RT.
Centrifuge again. and wash pellet with 70-75 % Ethanol.


You can increase the SDS percentage to facilitate Cell and tissue disrupting.
And for RNA extraction:
http://babakmemari.wordpress.com/2012/05/19/trizol-recipe-and-protocol/

-----
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