A sleazy way to avoid direct PCR Sequencing

John Altman altman at cmgm.stanford.edu
Fri Jan 21 12:36:15 EST 1994

> (Seth Findley) wrote:
> >
> >Now, rather than pick ONE colony for sequencing, I scraped about one 
> >*thousand* colonies off the plate and resuspended and mixed (well) the mass 
> >in a small volume of medium. Then I used a bit to inoculate an overnight 
> >with. I did an Acid-Phenol miniprep for sequencing and got nice clean 
> >sequence.  (I used an internal primer, so any vector subclones wouldn't 
> >be primed by my internal primer).
> >

In article <sk.1109553612A at inet>, sk at lilly.com (stuart kuhstoss) wrote:

> In that case you only need to worry if some
> (incorrect) sequence is favored over the correct one during growth.  That's
> possible, of course, but perhaps less likely.  
> Stu Kuhstoss

What's wrong with resuspending your thousand colonies (or many, many more Ñ
perhaps you could use large agar plates) in some media, spinning them down
and miniprepping directly, without growing overnight?  That completely 
takes care of the problem that Stu mentioned.

John Altman
altman at cmgm.stanford.edu

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