FW: crude bacterial DNA prep - a caution

Deanne Bell dbell at fresno.ars.usda.gov
Thu Aug 11 11:01:38 EST 2005


That's a good point!

Deanne Bell

 

>-----Original Message-----
>From: Dr. Hiranya S. Roychowdhury [mailto:hroychow at nmsu.edu] 
>Sent: Wednesday, August 10, 2005 2:14 PM
>Subject: Re: crude bacterial DNA prep - a caution
>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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