Direct PCR from E. coli colonies (GENOMIC???)

[Brad Nicholson]

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. 
YMMV.

Good Luck,
Brad 
===========================================================================
Brad Nicholson              |"If it worked the first time, it wouldn't be 
Department of Pathology     |	research."...Brad Nicholson
University of Utah          |	Live from behind the Zion Curtian.
Salt Lake City, UT 84132    | 
brad at corona.med.utah.edu    |
or: (801)-581-4365          | My opinions are solely my own.
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