crude bacterial DNA prep - a caution

Dr. Hiranya S. Roychowdhury hroychow at nmsu.edu
Wed Aug 10 16:14:09 EST 2005


Deanne and colleagues,  The colony PCR is a great way of minimizing handling 
and effort. PCR reactions can directly be supplemented by a portion of a single 
colony, using a sterile toothpick.  However, there is one caution:  This method 
often misleads with false poitives IF the colonies are off a plate that is the 
product of a fresh ligation event.

Hiranya.

Quoting Deanne Bell <dbell at fresno.ars.usda.gov>:

> Hey Michael
> 
> For PCRs I regularly use bacteria that have been scraped from media
> plates, suspended in water, aliquoted into utubes and stored in the
> freezer.  Just thaw an aliquote and use 1ul at 10e8 cells/ml in a 25ul
> PCR.  I would go ahead and try a PCR of I were you.  It is sooo much
> easier than doing the DNA prep.
> 
> I have also been using this really easy prep protocol published by Blake
> Bextine / Jacqueline Fletcher's lab using QBiogene's geneclean kits.  I
> believe the reference is something about yellow vine disease and
> associated bacterial infections of squash.  Plant Diseases 2001. Let me
> know if you want my protocol.
> 
> Deanne Bell
> USDA Agricultural Research Service
> Crop Diseases, Pests & Genetics
> 9611 South Riverbend Avenue
> Parlier, CA  93648
> voice  (559) 596-2806
> fax      (559) 596-2897
> dbell at fresno.ars.usda.gov
>  
>  
>  
> 
> >-----Original Message-----
> >From: methods-bounces at oat.bio.indiana.edu
> >[mailto:methods-bounces at oat.bio.indiana.edu] On Behalf Of Michael 
> >Sullivan
> >Sent: Monday, August 08, 2005 9:59 AM
> >To: methods at magpie.bio.indiana.edu
> >Subject: crude bacterial DNA prep
> >
> >I want to use PCR to confirm the presence of a large, low copy number 
> >plasmid from a bacterial strain.
> >
> >Can one simply use a small amount of culture directly in a PCR 
> >reaction, or should one do some sort of DNA prep, even a crude one, 
> >prior to PCR? If the latter, does anybody have any suggestions for an 
> >easy prep to make PCR-quality DNA.
> >
> >Thanks,
> >
> >Mike
> >---
> >Michael L. Sullivan
> >Plant Research Molecular Geneticist
> >US Dairy Forage Research Center
> >ARS-USDA
> >1925 Linden Drive West
> >Madison, WI 53706
> >(608) 890-0046 (Phone)
> >(608) 890-0076 (FAX)
> >
> >_______________________________________________
> >Methods mailing list
> >Methods at net.bio.net
> >http://www.bio.net/biomail/listinfo/methods
> >
> 
> 
> 
> 
> 
> 


-- 
Hiranya S. Roychowdhury, Ph.D.
Coll. Asst. Professor,
Molecular Biology,
Dept. of Chemistry & Biochemistry
Rm# 336, Chemistry Bldg.; MSC 3MLS
New Mexico State University
Las Cruces, NM 88003



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