AW: Methods Digest, Vol 39, Issue 2

Matthias Leiser via methods%40net.bio.net (by matthias.leiser from agrobiogen.de)
Wed Aug 6 00:04:58 EST 2008


 
Hi Wo,

Look to the link below, maybe this gives you an idea how to solve your
question.
Best regards
Matthias 

http://www.invitek.eu/content/forschung__technologie/technologien/molekulare
_diagnostik/index_ger.html
> is there a way how one can make the DNA polymerase (ordinary Taq 
> should do, no proofreading activity is desired) stop within a sequence 
> introduced by a synthetic primer? I thought of something like several 
> sugar building blocks without any base attached or a modified base 
> which is so bulky that the pol must stop and slip off.
> The modified position does not need to align to the parent DNA strand 
> as it may be placed in a loop. It just should be able to be 
> synthesized with standard phosphoramidite technology.
>
> Many thanks for sharing your thoughts,
>
> Wo
>



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Message: 2
Date: Mon, 04 Aug 2008 19:55:48 GMT
From: dk from no.email.thankstospam.net (DK)
Subject: Re: How to make polymerase stop in sequence?
To: methods from net.bio.net
Message-ID: <g77mpj$otg$1 from news.albasani.net>

In article
<05640717-17d1-41f8-bf28-657fe28c55f3 from y38g2000hsy.googlegroups.com>, WS
<novalidaddress from nurfuerspam.de> wrote:
>Dear Experts,
>
>is there a way how one can make the DNA polymerase (ordinary Taq should 
>do, no proofreading activity is desired) stop within a sequence 
>introduced by a synthetic primer? I thought of something like several 
>sugar building blocks without any base attached or a modified base 
>which is so bulky that the pol must stop and slip off.
>The modified position does not need to align to the parent DNA strand 
>as it may be placed in a loop. It just should be able to be synthesized 
>with standard phosphoramidite technology.

Polymerase normally pops off when it encounters already present strand. 
So, how about throwing in another primer that anneals specifically to the
sequence where you want the poly to stop? Having 3' phosphoramidite will
prevent synthesis from this "killer" primer. Or make the 3' base dideoxy.
Same thing. 

To prevent strand displacement, you can do extension with PfuUltra at 65C
(or even below). 

Because I know no chemistry, that's what I'd do :-)

DK




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Message: 3
Date: Mon, 4 Aug 2008 18:02:09 +0100
From: Nikolay Zenkin <n.zenkin from newcastle.ac.uk>
Subject: Imidazole and Amicon concentrators
To: "'methods from iubio.bio.indiana.edu'" <methods from magpie.bio.indiana.edu>
Message-ID:
	
<EMEW-k73I2A03551ee1dd21aff469eb1a9efe57eac2-33F9CC2CF4301E46874B36A6CB3E314
113F0D2DB21 from EXSAN02.campus.ncl.ac.uk>
	
Content-Type: text/plain; charset="us-ascii"

I used to concentrate by just ammonium sulphate precipitation with
subsequent dialysis.


Nikolay




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

Message: 4
Date: Tue, 5 Aug 2008 02:28:20 +0000
From: =?gb2312?B?sPzI8Q==?= <baorui_ren from hotmail.com>
Subject: HELP! protein-protein cross-linking
To: <methods from magpie.bio.indiana.edu>
Message-ID: <BLU146-W195971D31B5E8B8C0EB161EB7B0 from phx.gbl>



Hello, AllWe have a problem in the chemical cross-linking experiment for
protein-protein interaction. We have solved a protein complex structure, and
we want to confirm their interaction by cross-linking. But i am a new comer
in this field, the initial test was failed. Protein A is 35kd, Protein B is
14kd, but after cross-linking the band representing 49kd is very very weak
in our SDS-PAGE comparing with the inact Protein A and Protein B in thesame
lane. The condition we used is : add DSS to protein sample containing
protein A and protein B and incubated for 1 hour under 4 degree, and then
terminated it with 1M tris for 30 min. The samples were loaded on SDS-PAGE.
We have tried different concentrations of DSS (0.1mM-10mM) and protein
sample (20uM-1mM). PS: from the complex structure , we have found several
Arg residues in both protein were located in the interface, so we use the
DSS for initial test.Protein A trends to form dimer, but we could not even
got the dimer bands af!
 ter cross-linking. I would like to hear any suggestion from you.Thanks!




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