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Is there an optimal incubation time for cell lysis?


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#1 Wolverena

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Posted 25 February 2009 - 07:30 AM

I have been looking through many cell lysis protocols over the last months and I noticed a huge variety of incubations times although most protocols had the same enzymes or detergents. Granted, some were for pure grown cultures and others for environmental samples, but I was wondering why the incubation times are so different (with such a wide range). For example, there were protocols for enzymatic lysis with lysozyme ranging from 30min to several hours and for detergent (SDS or CTAB) from 30 min to several hours (or even overnight). Well, 30 min versus 6 hours is quite a difference.....which one is better? Is it better to be on the "save side" and have longer incubation times or could it damage the DNA (when using cell lysis for DNA extractions)? What do you think?

Cheers!
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#2 molgen

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Posted 25 February 2009 - 07:47 AM

I guess that it depends on the cell type.
You can always try to redoes the time and see if it changes anything.

From my experience with CTAB usually 30 min. were enough.

#3 aimikins

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Posted 25 February 2009 - 09:18 AM

I agree. it absolutely depends on cell type, in terms of the membrane structure. the optimal lysis protocol will break down the cell wall or cell membrane without destroying whichever internal structures you desire to keep. it all depends on the downstream application, as well.

for example, lying a bunch of S aureus cells requires a pretty stringent protocol because of the all the peptidoglycan. lysing mammalian TC cells while keeping the nuclei intact for a cytoplasmic extract would be much more gentle and delicate, do you see?
"it is a miracle that curiosity survives formal education" -A.E.

#4 Wolverena

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Posted 25 February 2009 - 09:29 AM

yeah, I see your point. Thanks guys for your input.

another question: let's say you have a mixed cell sample, such as an environmental sample (water or soil) where you don't know what's in there.....how would you handle that one?
"You can give somebody a book on 'How to ride bike' and then test that person on that knowledge. Even if that person gets an "A", it doesn't mean that he or she can ride a bike."
---this is what I am telling myself when I get a bad grade....as long as you don't loose your passion, you'll be fine.....V

#5 molgen

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Posted 26 February 2009 - 02:02 AM

For soil I'd add water, vortex, let stand for 10 min (so that the sediment will sink), move the supernatant to a new tube, spin, save the pellet and discard the sup.

For water I'd just spin and save the pellet like in the soil.

#6 aimikins

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Posted 26 February 2009 - 08:57 AM

again, what's your downstream application? are you looking for proteins? will there be a DNA prep? why are you lysing the cells?
"it is a miracle that curiosity survives formal education" -A.E.

#7 Wolverena

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Posted 26 February 2009 - 09:33 AM

again, what's your downstream application? are you looking for proteins? will there be a DNA prep? why are you lysing the cells?


DNA extractions, which than will be used for PCR (and phylogenetic analysis). I am using a clean up kit after the lysis, but I would like to make sure to get as much DNA as possible out of the sample. And incubation times for enzymatic lysis might play an important role in it.
"You can give somebody a book on 'How to ride bike' and then test that person on that knowledge. Even if that person gets an "A", it doesn't mean that he or she can ride a bike."
---this is what I am telling myself when I get a bad grade....as long as you don't loose your passion, you'll be fine.....V

#8 aimikins

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Posted 26 February 2009 - 02:51 PM

hmmm I would probably use a standard SDS / NaOH protocol, maybe also add some lysozyme, unless you think you have a bunch of gram positive bugs in your sample
"it is a miracle that curiosity survives formal education" -A.E.




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