I tried to use the freeze and thaw method but it's not working. My friend who work with same method did it as well, but his bacteria is from soil.
What is the actual problem? Do you not achieve cell lysis or does your DNA get destroyed in the process. I do not work with marine bacteria but if you do not achieve lysis you might want to try glas beads and a fast prep or vortex? Did you try Lysozyme?
We used a method including TCA for complete DNA isolation from Deep Sea bacteria In NIO, Goa. We then ran it on AGarose gel and cut out genomic DNA.
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2576689/
For vibrios we boil cultures that are in tubes for 10 minutes and then centrifuge at 10000 x g for 10 minutes to remove cellular components. Keep the supernatand containing the DNA. This is a very crude way to do it, but it works well of you need to do something simple like PCR using the DNA. For a more "clean" method you can probably use a DNA isolation kit, which you can buy from several places.
Did you run a gel with the results of the extraction? You can boil them also, use chloroform/phenol to get rid of the DNAses, or any other method. Try to see what is blocking the yield, breaking up the cells or DNA degradation.
Of course there should be some kits to do what you want, but if you want to do old school, you might have to adapt some of the classic techniquens. Take a look into Sambrook et. al. (molecular cloning)
You can try grinding in LN and adding some PVP. If the sample is just biomass, add some baked silica sand.
In many cases, the matrix of the sample is as much or even more important than the nature of the cells themselves at the time of choosing a DNA extraction method.
Are your bacteria from a water or a sediment sample?
If water, how do you collect them? Filtration? Centrifugation?
If sediments, where from? Are they from the anoxic layer, i.e. with black sulfide precipitates? Are they carbonate rich? Is it rich in clay-size fraction?
And in general.... How much sample do you use?
We had some issues with soil bacteria isolation and decided to go with the "throw money at it" solution to our problem. The kits from Mo-Bio have been super useful and we have had nice DNA preps every since selecting them.
http://www.mobio.com/water-sample-type/
They will send out free samples, so give it a try!
Thank you very much for all of yor help, but here i just try classic technique without Kit.
@Michaela & Mathias: I used the Lysis buffer
@Surabh: thank you for ur sharing
@Mesrop &gustavo : actually i used the freeze n Thaw method (thaw is boiling the sample but just 5 minute), thank you for remember me -->sambrook
@Stalis: thank you, i think my bacteria is Gram-positive
@Xabier: my bacteria is from water, to collect it with the Centrifugation, precipitates, i had 4 sample bacteria but just 1 bacteria it can't to genom isolated.
@David: seriously, is that really free?
Then you can try with some soil kits. The water kits are designed for processing water samples, but if you already have a pellet I'd go for a soil kit.
Most companies send kit samples for free
Yes, they really do send you two samples for free. I would recommend the kits the have bead beating or some sort of agitation feature and DNAse inhibitors.
FREE --> http://www.mobio.com/samples-all/powerwater-dna-isolation-sample-kit.html
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