Methods Digest, Vol 32, Issue 10

BAHRAM KAZEMI via methods%40net.bio.net (by bahram_14 from yahoo.com)
Mon Jan 14 00:30:47 EST 2008


I will clone and express the 16 KDa protein gene of echinococcus granolusus, but I don`t find its sequence in  gene bank ( echinococcus complete genome or mRNA of mentioned gene)
Bahram Kazemi

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Today's Topics:

   1. Re: high molecular plasmid DNA (WS)
   2. Re: high molecular plasmid DNA (Dr. Hiranya S. Roychowdhury)


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Message: 1
Date: Sat, 12 Jan 2008 02:56:51 -0800 (PST)
From: WS 
Subject: Re: high molecular plasmid DNA
To: methods from net.bio.net
Message-ID:
 <24f34919-398a-4921-87b7-2fc67b03d8a1 from j78g2000hsd.googlegroups.com>
Content-Type: text/plain; charset=ISO-8859-1

that means: make the alkaline lysis step in plasmid isolation as short
as possible! Some protocols state "5 min", but for standard lab
strains of E. coli that's complete overkill.

Add SDS/NaOH (lysis solution), mix shortly by snipping with your
fingers, put on ice, add neutralizing solution and mix again
immediately. Never vortex, as this would shear genomic DNA.

Best regards,

Wo


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Message: 2
Date: Sat, 12 Jan 2008 17:02:30 -0700 (MST)
From: "Dr. Hiranya S. Roychowdhury" 
---------------------------------

Subject: Re: high molecular plasmid DNA
To: aawara from pontiff-playground.org
Cc: methods from magpie.bio.indiana.edu
Message-ID: <2325.71.210.244.200.1200182550.squirrel from webmail.nmsu.edu>
Content-Type: text/plain;charset=iso-8859-1

This is a perfectly good explanation.  Thank you. Nicking of the plasmid
-as well as of the chromosomal pieces - does cause the HMW bands to
appear.  Another interesting observation is that often a clean and
perfectly good prep will show these spurious species on subsequent
electrophoresis (especially following storage).

> In ,
>  Dr. Hiranya S. Roychowdhury 
---------------------------------
 wrote:
>
>> Well, this has never been suficiently explained, nor do I know of any
>> specific investigation into this.  I tend to believe that the high MW
>> species may exist as a complex of bacterial chromosome and other
>> proteins.
>>  Usually, a clean plasmid prep does not show the HMW bands.  It also has
>> nothing to do with the conc. of the agarose gel.
>
> Excessive treatment with alkali will cause this to appear.  When nicked
> DNA denatures, it creates a fast-migrating single-stranded circle that
> is "undigestable" by restriction endonucleases.  The other product is
> linear ssDNA that tends to aggregate (by partial base-pairing) into
> large complexes that migrates near the well of agarose gels.
>
> If you treat minipreps with a single-strand specific nuclease like
> S1 or mung-bean nuclease both aberrant species (hmw ssDNA, and fast-
> migrating ssDNA) will disappear.
>
> AC
> --
> Email: echo 36434455860060025978157675027927670979097959886449930P | dc
> _______________________________________________
> Methods mailing list
> Methods from net.bio.net
> http://www.bio.net/biomail/listinfo/methods
>


-- 
Hiranya S. Roychowdhury, Ph.D.
Asst. Professor,
Health & Public Services
Dona Ana Community College
New Mexico State University
Las Cruces, NM 88003



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End of Methods Digest, Vol 32, Issue 10
***************************************



Bahram Kazemi Ph.D., Associate Professor
  1- Parasitology Department
  2- Cellular and Molecular Biology Research Center
  Shaheed Beheshti  University of Medical Sciences, Tehran, I.R. Iran 
PO Box: 19395 -4719 
Telefax: +98  21 22428432
url: www.bahramkazemi.ir 
E-mail:
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