pcr from E. coli colonies

4700gbera at umbsky.cc.umb.edu 4700gbera at umbsky.cc.umb.edu
Sat Jul 15 14:18:43 EST 1995


In article <3u6bio$fbv at news.cc.utah.edu>, brad at corona.med.utah.edu (Brad Nicholson) writes:
>In article <1995Jul13.114706.8906 at mbcf>, heath at mbcf.stjude.org 
>(Richard_Heath) wrote:
> >
> >Has anybody used this successfuly for amplifying a gene from *genomic* 
> >E. coli DNA?  If so, what kind of yield did you get from how many cycles?
> >(I'm thinking of trying it, but just want a few pointers first!)
> >
> >TIA
> >
> >Richard 
>
>Hello Richard,
>Our lab routinely amplifys genes from E. coli genomic DNA.  We have used 
>pure chromosomal DNA (CsCl pure, Qiagen), PCR kits (Bio-Rad Insta-Prep 
>matrix), and by the quick resuspend/boil methods that other people have 
>described in this thread.  Generally the success rate is higher with more 
>pure DNA, probably less salt or other nasties.  Cycles and yields are 
>dependent upon the primers and gene (size, GC content, etc.) amplified, but
>generally in my hands 30 cycles gives a decent yield ~ 500 -> 10,000 ng. 

We've had success just using E. coli colonies. We basically just pick up
a bit of colony with a pipet tip and mix it with the pcr cocktail. If you
use too much colony it won't work right, but after a couple of tries you get
the hang of it. 

-gina




















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