Low concentration miniprep

Utsugi, Heidi K via methods%40net.bio.net (by hutsugi from fhcrc.org)
Thu Jan 15 13:16:55 EST 2009


"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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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
Utrecht University
Krytgebouw
Padualaan 8
NL-3584 CH Utrecht
The Netherlands

office phone +31 30 253 4105
mobile phone +31(0)631154267

private address:
Griftkade 4 bis
3572 TW Utrecht

I never think of the future. It comes soon enough.
     Albert Einstein
Res severa verum gaudium. (~true delight is a severe issue)
     Seneca


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