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Methods Digest, Vol 17, Issue 23

Methods Digest, Vol 17, Issue 23 - Protocols and Methods Forum

Methods Digest, Vol 17, Issue 23 - Post Any Protocol, Method, Technique, Procedure or Tips / Troubleshooting for any Molecular Biology Technique.


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Old 10-25-2006, 09:11 AM
Sayyari, Mohammad
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Default Methods Digest, Vol 17, Issue 23



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

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Sent: Tue 10/24/2006 7:02 PM
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Subject: Methods Digest, Vol 17, Issue 23



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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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