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I have a urgent questuion,

I have a urgent questuion, - Protocols and Methods Forum

I have a urgent questuion, - Post Any Protocol, Method, Technique, Procedure or Tips / Troubleshooting for any Molecular Biology Technique.


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  #1  
Old 10-30-2006, 09:42 AM
Sayyari, Mohammad
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Default I have a urgent questuion,



Dear Sir/Madam

I am trying to measure oxidizer -reducer microorganism in rhizosphere soil.
Can one tell me the method? I would appreciate it, if you could help
me.

Best regards,
Mohammad



________________________________

From: [Only registered users see links. ] on behalf of
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Sent: Wed 10/25/2006 7:01 PM
To: [Only registered users see links. ]
Subject: Methods Digest, Vol 17, Issue 24



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Today's Topics:

1. Re: Wessel-Flugge Method for Protein Precipitation (DK)
2. RE: Methods Digest, Vol 17, Issue 23 (Sayyari, Mohammad)


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

Message: 1
Date: Wed, 25 Oct 2006 02:06:28 GMT
From: [Only registered users see links. ] (DK)
Subject: Re: Wessel-Flugge Method for Protein Precipitation
To: [Only registered users see links. ]
Message-ID: <DEz%g.56$[Only registered users see links. ]>

In article <[Only registered users see links. ].net> , Jeremy Kamil
<[Only registered users see links. ]> wrote:

I don't know, it always worked extremely well for me. Beats TCA any
day. I always remove the aqueous layer as completely as I can -
basically, until the meniscus collapses. I also always spin at all steps
much harder than, IIRC, indicated (1 min at 12K). The protein is on
interphase, so it is not a good idea to remove it. Don't think it's
detergents causing it in your case, since I used to remove 1%
Triton X-100 with no problems. Maybe try using more chloroform
during the initial denaturing step?

DK



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

Message: 2
Date: Wed, 25 Oct 2006 11:11:58 +0200
From: "Sayyari, Mohammad" <[Only registered users see links. ]>
Subject: RE: Methods Digest, Vol 17, Issue 23
To: <[Only registered users see links. ].indiana.edu>
Message-ID:
<[Only registered users see links. ].to p.gwdg.de>
Content-Type: text/plain; charset="iso-8859-1"

Dear Sir/Madam

I am trying to measure oxidizer -reducer microorganism in rhizosphere soil.
Can one help me and tell the method? I would appreciate it, if you could help
me.

Best regards,
Mohammad

_____

From: [Only registered users see links. ] on behalf of
[Only registered users see links. ]
Sent: Tue 10/24/2006 7:02 PM
To: [Only registered users see links. ]
Subject: Methods Digest, Vol 17, Issue 23



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[Only registered users see links. ]
or, via email, send a message with subject or body 'help' to
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[Only registered users see links. ]

When replying, please edit your Subject line so it is more specific
than "Re: Contents of Methods digest..."


Today's Topics:

1. Re: low molarity borate and acetate agarose gels (Pow Joshi)
2. Re: Stripping Solution (Pow Joshi)
3. Wessel-Flugge Method for Protein Precipitation (Jeremy Kamil)
4. pEGAD,pGreenII and p35S-GFP-JFH1 (Jinxiang Wang)


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

Message: 1
Date: Mon, 23 Oct 2006 15:41:49 -0500
From: "Pow Joshi" <[Only registered users see links. ]>
Subject: Re: low molarity borate and acetate agarose gels
To: "Ho-Leung Ng" <[Only registered users see links. ]>
Cc: [Only registered users see links. ]
Message-ID:
<710764ea0610231341g7406872egc17c77adbc37d2a4@mail .gmail.com>
Content-Type: text/plain; charset=ISO-8859-1; format=flowed

Hi,

frankly, I had run, regularly, some TBE gels (tris borate EDTA) and
the protocol is available in the Molecular cloning book, especially
the old editions by Tom Maniatis.....
You could use 0.5X instead of 1x buffer; alternatively, you could use
low voltages.
I used to find the resolutions comparable .... although it's easy to
run TAE gels if you wish to isolate/ purify your DNA.

hope this helps

pow
On 10/19/06, Ho-Leung Ng <[Only registered users see links. ]> wrote:



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

Message: 2
Date: Mon, 23 Oct 2006 16:20:20 -0500
From: "Pow Joshi" <[Only registered users see links. ]>
Subject: Re: Stripping Solution
To: "Christian Praetorius" <[Only registered users see links. ]>
Cc: [Only registered users see links. ]
Message-ID:
<710764ea0610231420i24c17122y2e75e60b1e9674f0@mail .gmail.com>
Content-Type: text/plain; charset=ISO-8859-1; format=flowed

On 10/15/06, Christian Praetorius <[Only registered users see links. ]> wrote:

you might want to add some beta mercaptoethanol or DTT as well ...
this would cleave the -S-S- linkages in the antibodies (again assuming
you want to strip western blots)

pow



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

Message: 3
Date: Tue, 24 Oct 2006 02:29:19 -0400
From: Jeremy Kamil <[Only registered users see links. ]>
Subject: Wessel-Flugge Method for Protein Precipitation
To: [Only registered users see links. ]
Message-ID: <[Only registered users see links. ]>
Content-Type: text/plain; charset=US-ASCII; delsp=yes; format=flowed

Hi, I'm trying to concentrate dilute protein mixtures in my tandem
affinity purification eluates for mass spec analysis. Specifically, I
need to get rid of the EDTA and EGTA as well as the 0.1% tween or
NP-40 that is in my elution buffer.

I saw that some people use the Wessel-Flugge method (Anal Biochem
1974) for this application (Methanol, Chloroform precipitation).
However, in my hands I found it didn't seem to work great, at least I
don't see a nice pellet.

I've had some success in the past with TCA but often end up with some
residual acidity even after many acetone washes. Anyway, at least
with TCA I got a nice pellet every time.

My question re: Wessel-Flugge is, HOW MUCH OF THE UPPER LAYER ARE YOU
SUPPOSED TO REMOVE??? Should I get rid of the upper phase all the way
down to the chloroform layer, or leave a bit above of the chloroform??

The original publication says to remove the upper layer above the
chloroform and interphase. But I don't see much of an interphase,
except in some samples where I saw a diffuse band of white above the
chloroform layer on the bottom. so, in case that diffuse white stuff
was my protein, I left maybe 150 uL on top of the chloroform. In the
end I did not see much, if any, precipitate and was pretty
disappointed since the method seemed so promising.

any advice would be very much appreciated. I found the description by
Wessel and Flugge pretty cursory.

thanks,

Jeremy





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

Message: 4
Date: Tue, 24 Oct 2006 19:39:49 +0800
From: Jinxiang Wang <[Only registered users see links. ].cn>
Subject: pEGAD,pGreenII and p35S-GFP-JFH1
To: [Only registered users see links. ], [Only registered users see links. ]
Message-ID: <[Only registered users see links. ].cn>
Content-Type: text/plain; charset=GB2312

Hello,All,

I appreciate someone who would like to share pEGAD,pGreenII and
p35S-GFP-JFH1 with me to construct GFP-tagged plasmid to transform
/Arabidopsis/. Thank you in advance.

Jinxiang

--
Jinxiang Wang Ph.D
College of Resources and Environment
Root Biology Center
South China Agricultural Univeristy
Guangzhou 510642, P.R.China
Tel:0086-20-85280156
Fax:0086-20-85281829
Email:[Only registered users see links. ].cn



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  #2  
Old 10-30-2006, 05:50 PM
Duncan Clark
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Posts: n/a
Default I have a urgent questuion,

Historians believe that in newspost
<[Only registered users see links. ].ne t> on Mon, 30 Oct 2006,
"Sayyari, Mohammad" <[Only registered users see links. ]> penned the following literary
masterpiece:

I have no idea as it isn't my field but if I had to start I would a) do
a google or a.n.other www search engine search and b) do a Pubmed
search. Having found a paper or article that looks promising I would
then use better keywords to expand on the search and look closely at the
references in the paper.

The tools are out there to do this, it just takes a lot of reading to
find an answer or to find a more specific question to ask here.

Duncan
--
I love deadlines. I especially like the whooshing noise they make as
they go flying by.

Duncan Clark
GeneSys Ltd.
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