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Low concentration miniprep

Low concentration miniprep - Protocols and Methods Forum

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Old 01-15-2009, 06:16 PM
Utsugi, Heidi K
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Default Low concentration miniprep



"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"



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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" <[Only registered users see links. ]>
Subject: 2D gel analysis software
To: [Only registered users see links. ]
Message-ID:
<77ddf6c70901140044p545aed82q52a18dcb1e87dcbf@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: [Only registered users see links. ]


------------------------------

Message: 2
Date: Tue, 13 Jan 2009 17:04:38 -0800 (PST)
From: muhammad yasir <[Only registered users see links. ]>
Subject: Low concentration miniprep
To: [Only registered users see links. ]
Message-ID: <[Only registered users see links. ].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" <[Only registered users see links. ]>
Subject: Re: Low concentration miniprep
To: [Only registered users see links. ]
Cc: [Only registered users see links. ]
Message-ID:
<bd2597a20901150346m1f5cbb11h369a0043b2034be7@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 <[Only registered users see links. ]>




--
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 <[Only registered users see links. ]>
Subject: Black hole quenchers (BHQ) and 3' blocking in Real-time PCR
To: [Only registered users see links. ]
Message-ID:
<[Only registered users see links. ]>
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" <[Only registered users see links. ]>
Subject: Re: Low concentration miniprep
To: "Fulvio Celsi" <[Only registered users see links. ]>
Cc: [Only registered users see links. ], [Only registered users see links. ]
Message-ID:
<123243900901150741jaa1901p96f0c9f261b153fe@mail.g mail.com>
Content-Type: text/plain; charset=UTF-8

Hi Yasir


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




--
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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