Methods Digest, Vol 50, Issue 15-Cultures

[Virash Gupta]

For XL-1-Blue use Tetracycline 5 microgram/ml.

On 7/22/09, methods-request from oat.bio.indiana.edu <
methods-request from oat.bio.indiana.edu> wrote:
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> Today's Topics:
>
>   1. Re: [a bit OT] floating "precipitates" (DK)
>   2. Re: protein extraction (Dr Engelbert Buxbaum)
>   3. Re: [a bit OT] floating "precipitates" (Dr Engelbert Buxbaum)
>   4. What is the working concentration of
>      N-succinyl-(Ala)3-Nitroanilide    when doing Elastase assay (feng
> zeng)
>   5. Re: TEV (DK)
>   6. Re: [a bit OT] floating "precipitates" (Pow Joshi)
>   7. Cultures (kamalaker nasani)
>
>
> ----------------------------------------------------------------------
>
> Message: 1
> Date: Tue, 21 Jul 2009 13:20:22 GMT
> From: dk from no.email.thankstospam.net (DK)
> Subject: Re: [a bit OT] floating "precipitates"
> To: methods from net.bio.net
> Message-ID: <rgj9m.33577$qM4.30204 from newsfe01.iad>
>
> In article <
> bca801ba-22dd-48d9-9073-ae4fb9dafd18 from a7g2000yqk.googlegroups.com>, WS <
> novalidaddress from nurfuerspam.de> wrote:
> >Dear Experts,
> >
> >please excuse my ignorance: How to describe stuff that goes out of
> >solution, but does not precipitate at the bottom of the vial and
> >prefers floating atop of it - Floating insoluble matter? Flotulate?
>
> I think it's flocculate.
>
> DK
>
>
> ------------------------------
>
> Message: 2
> Date: Tue, 21 Jul 2009 09:23:28 -0400
> From: "Dr Engelbert Buxbaum" <engelbert_buxbaum from hotmail.com>
> Subject: Re: protein extraction
> To: methods from net.bio.net
> Message-ID: <op.uxe55ee066vu6s from bengelbert-dm.rusm.rossu.loc>
> Content-Type: text/plain; format=flowed; delsp=yes;
>        charset=iso-8859-15
>
> Am 19.07.2009, 16:47 Uhr, schrieb riccardo senter <ricc_wparma from yahoo.it>:
>
> > hi!
> > i'm a new entry.
> > i need to extract proteins from PBMC in order to perform a western blot
> > and a functional assay of iNOS, which is a cytosolic homodimeric protein.
> > which is the best extraction protocol?
> >
> > Another question: can i use PBMC pellets that have been storing for
> > several years at -20°?
>
> I would simply start with a hypotonic buffer (20 mM HEPES-KOH pH 7 or the
> like) with the usual protease inhibitor coctail and pass the cells through
> a fine (27G) needle a couple of times. Then spin down nuclei, membranes
> and the like in an Eppendorf centrifuge (refrigerated). 2 min on full
> speed should do it. The supernatant contains the soluble proteins.
>
> If that does not work, you need to add detergents to your buffer. A very
> effective way is to use CTAB, then use cationic electrophoresis and
> eastern blotting for detection (Anal. Biochem. 314 (2003) 70-6). CTAB is
> even more effective in solubilisation than SDS, yet at the same time so
> gentle that it is often possible to run zymograms.
>
> As to the old samples, if they have been prepared with protease
> inhibitors, snap-frozen, constantly stored at low temperatures (no
> self-defrosting fridges!) and if you want to detect only protein, not
> post-translational modification or activity, it should probably work,
> especially if you have samples of known target concentration stored in the
> same way as controls.
>
>
> ------------------------------
>
> Message: 3
> Date: Tue, 21 Jul 2009 09:28:13 -0400
> From: "Dr Engelbert Buxbaum" <engelbert_buxbaum from hotmail.com>
> Subject: Re: [a bit OT] floating "precipitates"
> To: methods from net.bio.net
> Message-ID: <op.uxe6dbo666vu6s from bengelbert-dm.rusm.rossu.loc>
> Content-Type: text/plain; format=flowed; delsp=yes;
>        charset=iso-8859-15
>
> Am 21.07.2009, 04:43 Uhr, schrieb WS <novalidaddress from nurfuerspam.de>:
>
> > please excuse my ignorance: How to describe stuff that goes out of
> > solution, but does not precipitate at the bottom of the vial and
> > prefers floating atop of it - Floating insoluble matter? Flotulate?
>
> "Precipitate" refers to the material that has become insoluble during a
> process. Whether the density of that material is higher or lower than that
> of the solution has nothing to do with it.
>
> If the material has a higher density than the solution, and is allowed to
> settle (possibly with the aid of a centrifuge) you get a "pellet".
>
>
> ------------------------------
>
> Message: 4
> Date: Tue, 21 Jul 2009 09:47:06 -0400
> From: feng zeng <fengzeng0827 from gmail.com>
> Subject: What is the working concentration of
>        N-succinyl-(Ala)3-Nitroanilide  when doing Elastase assay
> To: Methods from magpie.bio.indiana.edu
> Message-ID:
>        <2a82b3da0907210647u7991e3f2jc626dc6062494dd from mail.gmail.com>
> Content-Type: text/plain; charset=ISO-8859-1
>
> Hi,
>
> Do any people know what is the work concentration of
> N-succinyl-(Ala)3-Nitroanilide when doing Elastase assay?
> And what will be the colour when this substrate reacts with Elastase?
>
> I use the concentration 4.4mM, and and 20ul to make finally 200ul mixture
> of
> Elastase, Buffer and Substrate, and then measure the OD at 410nm
> I did not see good result:(
>
> Thank you all
> Feng
>
>
> ------------------------------
>
> Message: 5
> Date: Tue, 21 Jul 2009 15:23:58 GMT
> From: dk from no.email.thankstospam.net (DK)
> Subject: Re: TEV
> To: methods from net.bio.net
> Message-ID: <h44mhd$45o$2 from news.albasani.net>
>
> In article <mailman.623.1248113365.21502.methods from net.bio.net>, "Vicky
> Cook" <vcook from wfubmc.edu> wrote:
> >I wanted to clarify the DTT: you mean
> >use TCEP instead of DTT, or the other way around?
>
> Yes, use TCEP instead of DTT.
>
> DK
>
>
> ------------------------------
>
> Message: 6
> Date: Tue, 21 Jul 2009 17:06:52 -0400
> From: Pow Joshi <pow.joshi from gmail.com>
> Subject: Re: [a bit OT] floating "precipitates"
> To: DK <dk from no.email.thankstospam.net>
> Cc: methods from magpie.bio.indiana.edu
> Message-ID:
>        <710764ea0907211406g4f0b2ad1l722e2cb38336770c from mail.gmail.com>
> Content-Type: text/plain; charset=ISO-8859-1
>
> awesome.. that's the one .... so I checked the origin on OED, and they say
> the origin of the word is "flocculus" or a flock of wool....thanks Dima
>
> 2009/7/21 DK <dk from no.email.thankstospam.net>
>
> > In article <
> > bca801ba-22dd-48d9-9073-ae4fb9dafd18 from a7g2000yqk.googlegroups.com>, WS <
> > novalidaddress from nurfuerspam.de> wrote:
> > >Dear Experts,
> > >
> > >please excuse my ignorance: How to describe stuff that goes out of
> > >solution, but does not precipitate at the bottom of the vial and
> > >prefers floating atop of it - Floating insoluble matter? Flotulate?
> >
> > I think it's flocculate.
> >
> > DK
> > _______________________________________________
> > Methods mailing list
> > Methods from net.bio.net
> > http://www.bio.net/biomail/listinfo/methods
> >
>
>
> ------------------------------
>
> Message: 7
> Date: Wed, 22 Jul 2009 16:05:55 +0530
> From: kamalaker nasani <agbiok4 from gmail.com>
> Subject: Cultures
> To: methods from magpie.bio.indiana.edu,     Plant Tissue Culture
>        <PLANT-TC from lists.umn.edu>,       plantbio from magpie.bio.indiana.edu
> Message-ID:
>        <f5beaffc0907220335q732b179t51a2f20742a158c8 from mail.gmail.com>
> Content-Type: text/plain; charset=ISO-8859-1
>
> Hi,
>
> I have to use XL1 BLUE
>
> Just I want to know the difference in terms of transformation efficiency
> and
> protocol.
>
> What antibiotic we need to use the XL1 BLUE
>
> --
> Kamalaker Nasani,
> Biotechnologist,
> Global Transgenes Ltd.
>
>
> ------------------------------
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> End of Methods Digest, Vol 50, Issue 15
> ***************************************
>



-- 
Dr V K Gupta
Sr Microbiologist (Molecular Biology)
Insect Molecular Biology Lab
Department of Entomology
Punjab Agricultural University
Ludhiana (Pb)-141004- India
M: 09815963210