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how do you inactivate the enzymes after digestion? - (Nov/28/2005 )

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If I needed to remove an enzyme, I'd probably use a Qiagen spin column, like their PCR purification kit -- quick and reliable.


QUOTE (aimikins @ Nov 29 2005, 11:38 AM)
that's usually how I do it; I was just wondering if it were possible to save the time ...but, as we're only talking a few hours it's not significant....I was just curious mostly

i was wondering the question too,i had ETOH precipitated my digested DNA samples as you said,but the result wouldn't come forth until several days later.


I always PCI/EtOH precipitate. If anyone knows whether a plain EtOH precipitation will get rid of Taq (which I don't think it will, but who knows), let me know. Phenol and chloroform is not pleasant!



i am just too scared to precipitate DNA and so prefer gel purify my samples.

These days kits are there, which just takes 20-30 minutes per sample. thsi way, u get rid of junkie stuff and doesnt loose DNA, infact get lil concentrtaed in few microlitres. and i guess these kits r not that expensive too !!



Spin columns take 3-5 minutes... tongue.gif


well i think columns are quick too. But how to you get rid of your short fragment digested by this way?...


For instance the qiaquick columns have a cutt off value of about 100 bp or so. The way I see it, is that shorter DNA does bind to the membrane, but easily gets washed away, because (due to its smaller size) it doesn't bind too strong. Very long fragments, more than 10 kb, will bind too hard to get off.

This is just the way I look at it, I might be completely missing some other key point and what I've written might be completely wrong...


Sorry I was unclear...

I always gel purify digested PCR fragments before cloning -- single digests, double digests, it doesn't matter -- all of them get gel purified. I've found it saves me much more time and frustration at the "screen the colonies" stage than it costs me at the "prepare to ligate" stage.

The conversation seemed to have drifted over to "how to get rid of an enzyme?", and if I were just concerned with removing an enzyme (like the first enzyme in an incompatible double digestion or someting), then I'd use a spin column.


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