help with hiperthermophiles - cant break them for DNA extraction (May/03/2007 )
hi, i was wondering if anyone here works with hyperthermophiles. ive got a big problem and i kinda need some help. I CANT BREAK MY CELLS! And of course, that means i can't extract DNA. These critters are UV and autoclave resistant. I suspect they wont have much trouble with gamma radaition either.
I've tried everything i can think of:
- liquid nitrogen
- glass beads
- combinations of all those above
i come from a poor lab in a poor country, and a french press is outta the question here... ive already thought of that but i cant get my hands on one!
so... anyone have any ideas? please? my boss wont get off my back!
If you only need a gen or some gen from the organism you might try this:
I needed a gen from a thermophil once. I just put some of the culture (20 µl) in the microwaveoven. 2 min full power (600W). Then I added 20 µl water and did different dilutions of it and performed PCR. I got the gen cloned quite easy
Hmm... problem is that the hiperthermophyle cell membrane is really harder to brake (saturated + eter bond instead of usually ester bond)
Of all that you tried , maybe the only thing it could work is phenol because it's an unpolar solvent, and that is what you need because you're dealing with fat acids.. Have you tried standard phenol/chloroform extraction DNA protocol? And maybe using some detergents would be helpful....
agree with mujo,
try to use non-polar solvents
i don't know if lipase can breakdown the membrane