DNA extraction from really wierd microbes - (Oct/02/2006 )
ok, i cant say very much cuz i have a "no talking" policy here at work, but i'm really going outta my mind here... i have some hyperthermophiles which WILL NOT DIE! All I want is their DNA for a lousy 16S PCR...
lets see... they love miliQ water, freezing, boiling, sonication, all types of detergent, electricity, etc...
and acid, of course kills them, along with their DNA....
soooo... does anyone have any protocol around there for these types of extremophiles??? 
your suggestions are my salary 
lets see... they love miliQ water, freezing, boiling, sonication, all types of detergent, electricity, etc...
and acid, of course kills them, along with their DNA....
soooo... does anyone have any protocol around there for these types of extremophiles???
your suggestions are my salary
This is quite interesting. Sorry I don't work on extremophiles and thus know absolutely nothing.
However I was wondering, have you tried a guanidine thiocyanate based lysis solution?
NaOH+detergent+chaotropic agent (guanidine thiocynate) + phenol? Maybe even mix in some CTAB (if there are lots of polysaccarides)?
I know.... this is my answer to everything. But I am just such a fan of variations of this lysis solution. I fervently believe it is the cure to all ailments. (Failing that, a version of the basic formulation)
I was also wondering if you have considered soaking the little critters in somekind of organic solvent like dichloromethane or diethyl ether? They can't be that extremophilic to survive that. I know the DNA will be okay in those two solvents.
sounds like a nice idea... any idea of the concentration used? and how long to soak em? 
thnx
I'm assuming you've mined the literature already? what about contacting an author that also works on archaea, and asking for their protocol?
Maybe I've misunderstood the issue here but surely if you get your liquid culture of these thermophiles and perform a phenol extraction on them that would be just fine.
Nobody likes phenol in the face - FACT.
Ask someone who cuts up mice, and scale it down to cut up those "never say die" bugs.
kill them with electricity??
my electropoarator is very gud at that.
Nobody likes phenol in the face - FACT.
jajaja... i thought the same thing.... but unfortunately, they do
thnx
No idea really. I would try 75% diethyl ether and 25% dichloromethane and maybe a little water, and sonicate. But I guess it would be an experiment to try and find out the right conditions.
Lastly, another weird idea.... immerse said bugs in liquid nitrogen and then grind with a mortar and pestle. It would take a lot of grinding, but surely the cells would break under such physical attack.
sounds like a nice idea... any idea of the concentration used? and how long to soak em?
thnx
Lastly, another weird idea.... immerse said bugs in liquid nitrogen and then grind with a mortar and pestle. It would take a lot of grinding, but surely the cells would break under such physical attack.
Yes, this was my next suggestion - liquid nitrogen and French Press.
That should do it...should.
nice, nice... c'mon mjolner... soak up all these ideas.... about that french press, any alternatives to making one? remember here in the third world techonology is expensive and rare 
as soon as i get this ill post it