FW: crude bacterial DNA prep - a caution
dbell at fresno.ars.usda.gov
Thu Aug 11 11:01:38 EST 2005
That's a good point!
>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.
>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
>> 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
>> >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
>> >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.
>> >Michael L. Sullivan
>> >Plant Research Molecular Geneticist
>> >US Dairy Forage Research Center
>> >1925 Linden Drive West
>> >Madison, WI 53706
>> >(608) 890-0046 (Phone)
>> >(608) 890-0076 (FAX)
>> >Methods mailing list
>> >Methods at net.bio.net
>Hiranya S. Roychowdhury, Ph.D.
>Coll. Asst. Professor,
>Dept. of Chemistry & Biochemistry
>Rm# 336, Chemistry Bldg.; MSC 3MLS
>New Mexico State University
>Las Cruces, NM 88003
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