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maxiprep help - (Sep/20/2005 )

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It's okay to disagree -- I don't mind; and I know I'm probably of the minority opinion here.

But, I do have twenty years experience at routine DNA work -- cloning, PCR, Southerns, restriction digests, sequencing (about three-quarters of a million bases so far), etc. -- and I've never found it necessary to spec DNA. In fact, we don't even own such a spec... I've worked on three separate organisms (B. anthracis -- a Gram positive, P. aeruginosa -- a Gram negative, and B. fragilis -- a Gram negative anaerobe), and of course use the same E. coli host strains and vectors as everyone else (where possible), so it's unlikely to be an organism-specific thing...

The reason I even said anything about it is because macrosky, who started this thread, has now purified his or her DNA four times, and has assumed it "failed" because of a spec reading. Most likely, I would have done it once, proceeded with the cloning or whatever, and have already gotten the clone I wanted. Perhaps ignorance is bliss, but I bet I'd have my clone already... biggrin.gif

As I said in my prior post I've seen way too many people get hung up on such things and waste weeks trying to overcome them, when in my experience, they could've already moved on but for a number they're chasing.


There is a lot of error rate in the spec readings, so I can understand what you are saying, especially since most machines that I have seen are 20 years old and still using 5 1/4 floppy disks!


I agree that for the most part (PCR, Restriction etc.), you don't really need such pure DNA. You do any reaction after which you will clean it up again before proceeding anyway, so as long as the first thing you do with it works, there shouldn't be a problem.

If however you want to transfect cells, you better have high quality/pure DNA (my own experience).


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