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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