Low concentration miniprep

shifali chatrath via methods%40net.bio.net (by shifalich from rediffmail.com)
Sat Jan 17 01:45:43 EST 2009


  Just a query! Are satellite colonies correctly defined as slow growing colonies or a daugther colony of nearby parent colony?

Additionally, I once picked up satellite colonies, they never grew in LB medium.

HOwever, Yasir can try increasing the volume of medium for inoculation and getting a bigger pellet,keeping all other solutions' volume to be the same.
Shifali


On Thu, 15 Jan 2009 Utsugi,Heidi K wrote :
>"I have clone the gene in pUC19 and get conformation with restriction digestion. But unfortunately, I get very low quantity of plasmid after miniprep. While the control (circular pUC19) concentration is quite enough with miniprep kit that I used. I shall be very glad to know about the possible reason of low concentration and how can I improve the yield of clone plasmid from miniprep.
>
>Yasir"
>
> >From what I have seen, more often that not, overloading a commercial DNA miniprep column is very likely.  This may be copy number related. The next culprit is overgrown plates with satellite colonies picked instead of transformed. -- Heidi
>
>
>________________________________
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>To: methods from magpie.bio.indiana.edu
>Subject: Methods Digest, Vol 44, Issue 13
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>Today's Topics:
>
>    1. 2D gel analysis software (yoginee budhkar)
>    2. Low concentration miniprep (muhammad yasir)
>    3. Re: Low concentration miniprep (Fulvio Celsi)
>    4. Black hole quenchers (BHQ) and 3' blocking in Real-time PCR
>       (chovek69)
>    5. Re: Low concentration miniprep (David-Paul Minde)
>
>
>----------------------------------------------------------------------
>
>Message: 1
>Date: Wed, 14 Jan 2009 14:14:39 +0530
> From: "yoginee budhkar" <eenigoy from gmail.com>
>Subject: 2D gel analysis software
>To: methods from magpie.bio.indiana.edu
>Message-ID:
>         <77ddf6c70901140044p545aed82q52a18dcb1e87dcbf from mail.gmail.com>
>Content-Type: text/plain; charset=ISO-8859-1
>
>Hello!
>
>What exactly should one look for while selecting a particular 2D gel
>analysis software? Can people share their experiences about the soft wares
>they are using?
>
>Regards,
>-- Yg
>--
>Yoginee Budhkar
>Junior Research Fellow
>Food Engineering and Technology Department
>Institute of Chemical Technology
>Matunga, Mumbai 400019
>Contact: yogineeb.foodbio from udct.org
>
>
>------------------------------
>
>Message: 2
>Date: Tue, 13 Jan 2009 17:04:38 -0800 (PST)
> From: muhammad yasir <yasirphr from yahoo.com>
>Subject: Low concentration miniprep
>To: methods from oat.bio.indiana.edu
>Message-ID: <161483.83541.qm from web55906.mail.re3.yahoo.com>
>Content-Type: text/plain; charset=iso-8859-1
>
>I have clone the gene in pUC19 and get conformation with restriction digestion. But unfortunately, I get very low quantity of plasmid after miniprep. While the control (circular pUC19) concentration is quite enough with miniprep kit that I used. I shall be very glad to know about the possible reason of low concentration and how can I improve the yield of clone plasmid from miniprep.
>
>Yasir
>
>
>
>
>------------------------------
>
>Message: 3
>Date: Thu, 15 Jan 2009 12:46:31 +0100
> From: "Fulvio Celsi" <fulvio.celsi from gmail.com>
>Subject: Re: Low concentration miniprep
>To: yasirphr from yahoo.com
>Cc: methods from oat.bio.indiana.edu
>Message-ID:
>         <bd2597a20901150346m1f5cbb11h369a0043b2034be7 from mail.gmail.com>
>Content-Type: text/plain; charset=ISO-8859-1
>
>Hi Yasir
>is your gene very large?what kit are you using? if the gene (and so the
>plasmid) is large, maybe you should try to increase the quantity of the
>bacteria when doing miniprep...
>
>2009/1/14 muhammad yasir <yasirphr from yahoo.com>
>
> > I have clone the gene in pUC19 and get conformation with restriction
> > digestion. But unfortunately, I get very low quantity of plasmid after
> > miniprep. While the control (circular pUC19) concentration is quite enough
> > with miniprep kit that I used. I shall be very glad to know about the
> > possible reason of low concentration and how can I improve the yield of
> > clone plasmid from miniprep.
> >
> > Yasir
> >
> >
> >
> > _______________________________________________
> > Methods mailing list
> > Methods from net.bio.net
> > http://www.bio.net/biomail/listinfo/methods
> >
>
>
>
>--
>Fulvio Celsi
>BsC,PhD
>Sector of Neurobiology,
>International School for Advanced Studies, Scuola Internazionale di Studi
>Superiori Avanzati (SISSA),
>Trieste
>Italy
>
>Mobile: +393286489131
>
>
>------------------------------
>
>Message: 4
>Date: Thu, 15 Jan 2009 00:08:59 -0800 (PST)
> From: chovek69 <ivanoov from gmail.com>
>Subject: Black hole quenchers (BHQ) and 3' blocking in Real-time PCR
>To: methods from net.bio.net
>Message-ID:
>         <a0a35eb6-50d6-48bb-8eac-d98660c335f6 from n10g2000vbl.googlegroups.com>
>Content-Type: text/plain; charset=ISO-8859-1
>
>Dear experts,
>
>I am relatively new in the Real-time PCR world.  I am about to order a
>bunch of hydrolysis probes quenched with the BHQ.
>
>The company states exclusively that they can do 3' - BHQ labeling but
>is this also mean that the 3'-OH will be blocked by the quencher
>against extension  or I must order 3' phosphorylation in addition.
>
>Any suggestions will be most welcome...
>Thanks
>Ivan
>
>
>------------------------------
>
>Message: 5
>Date: Thu, 15 Jan 2009 16:41:28 +0100
> From: "David-Paul Minde" <davidminde from gmail.com>
>Subject: Re: Low concentration miniprep
>To: "Fulvio Celsi" <fulvio.celsi from gmail.com>
>Cc: methods from oat.bio.indiana.edu, yasirphr from yahoo.com
>Message-ID:
>         <123243900901150741jaa1901p96f0c9f261b153fe from mail.gmail.com>
>Content-Type: text/plain; charset=UTF-8
>
>Hi Yasir
>
> > is your gene very large?what kit are you using? if the gene (and so the
> > plasmid) is large, maybe you should try to increase the quantity of the
> > bacteria when doing miniprep...
>
>e.g. Terrific Broth would help in this case (and if still necessary) bigger
>volumes of culture.
>Of course you shold not exceed the specs of your kit (or you will have
>decrease of DNA quality)
>best,
>David
>
> >
> >
> > 2009/1/14 muhammad yasir <yasirphr from yahoo.com>
> >
> > > I have clone the gene in pUC19 and get conformation with restriction
> > > digestion. But unfortunately, I get very low quantity of plasmid after
> > > miniprep. While the control (circular pUC19) concentration is quite
> > enough
> > > with miniprep kit that I used. I shall be very glad to know about the
> > > possible reason of low concentration and how can I improve the yield of
> > > clone plasmid from miniprep.
> > >
> > > Yasir
> > >
> > >
> > >
> > > _______________________________________________
> > > Methods mailing list
> > > Methods from net.bio.net
> > > http://www.bio.net/biomail/listinfo/methods
> > >
> >
> >
> >
> > --
> > Fulvio Celsi
> > BsC,PhD
> > Sector of Neurobiology,
> > International School for Advanced Studies, Scuola Internazionale di Studi
> > Superiori Avanzati (SISSA),
> > Trieste
> > Italy
> >
> > Mobile: +393286489131
> > _______________________________________________
> > Methods mailing list
> > Methods from net.bio.net
> > http://www.bio.net/biomail/listinfo/methods
> >
>
>
>
>--
>David Minde MSc (TUM)
>
>Cellular Protein Chemistry Room 707
>Department of Chemistry
>Faculty of Science
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>      Albert Einstein
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>      Seneca
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Shifali Chatrath
Graduate Student
Protein science Lab
Dept. of Biological sciences
National University of Singapore
Singapore
+65-65161210
+65-96393449


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