Methods Digest, Vol 30, Issue 14-Bacterial viability assay

Virash Gupta via methods%40net.bio.net (by virashkgupta from gmail.com)
Thu Nov 22 23:21:32 EST 2007


Bacterial viability assay:
You need to write specific experiment-name of bacteria, cultural
conditions and what you want to test, for correct suggestions.

V K Gupta
Sr Microbiologist
Insect Mol Biology Lab
PAU

On 11/22/07, methods-request from oat.bio.indiana.edu
<methods-request from oat.bio.indiana.edu> wrote:
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> Today's Topics:
>
>   1. troubleshooting + ligation (Anajli Thomas)
>   2. bacterial viability assay (Sylvie Chauvaux)
>   3. Protein/protein-crosslinkers (Peter Henriksen)
>   4. RE: Protein/protein-crosslinkers (Deitiker, Philip R)
>   5. Re: troubleshooting + ligation (Sudheendra Rao N R)
>   6. Re: Protein/protein-crosslinkers (Sudheendra Rao N R)
>   7. Re: troubleshooting + ligation (Jose de las Heras)
>   8. cccDNA preparation from transfected HepG2 cells (WANG XF)
>
>
> ----------------------------------------------------------------------
>
> Message: 1
> Date: Wed, 21 Nov 2007 10:51:04 +0530
> From: "Anajli Thomas" <anjalirthomas from gmail.com>
> Subject: troubleshooting + ligation
> To: methods from magpie.bio.indiana.edu
> Message-ID:
>        <efb783e60711202121n42a43a6ft9370297f10335e30 from mail.gmail.com>
> Content-Type: text/plain; charset=ISO-8859-1
>
>      I have been trying to clone a 1.5 kb insert to pEGFP c1 vector. The
> restriction enzymes used were EcoR1 and Kpn1. I have tried ligation thrice
> and still no colonies. JM109 was used for transformation.
>
> The restriction enzymes were added together for cutting the insert from T
> vector (primary clone). For ligation I added 5 ul of insert and 2ul of
> backbone.
>
> Could you help me out with this problem
>
> Sincerely
> Anjali
>
>
> ------------------------------
>
> Message: 2
> Date: Wed, 21 Nov 2007 17:08:41 +0100
> From: Sylvie Chauvaux <chauvaux from pasteur.fr>
> Subject: bacterial viability assay
> To: methods from magpie.bio.indiana.edu
> Message-ID: <f06230905c36a04a2b13d@[157.99.29.21]>
> Content-Type: text/plain; charset="us-ascii" ; format="flowed"
>
> Hello,
>
> Does somebody know an assay to follow bacterial viability of a
> culture ? The commercial kits implicate to take samples of cultures.
> Since I need to follow the viability overnight, I need an assay where
> the reagents can be present in the culture without affecting growth
> of bacteria.
>
> Many thanks for all your ideas,
>
> Sylvie
>
>
>
> ------------------------------
>
> Message: 3
> Date: Wed, 21 Nov 2007 16:40:31 +0100
> From: "Peter Henriksen" <peterh from my.molbio.ku.dk>
> Subject: Protein/protein-crosslinkers
> To: <methods from magpie.bio.indiana.edu>
> Message-ID:
>        <B32B87E82F289941B2A4B7CAAF0F151A4DC3F9 from MAILSERVER.akizci.ku.dk>
> Content-Type: text/plain;       charset="iso-8859-1"
>
> I am trying to design an experiment in which I will need to make protein-protein crosslinks but not DNA-protein crosslinks in living cells.
>
> In principal I think this should be possible using a crosslinker that will crosslink between two carboxylic groups but I haven't been able to find such a crosslinker so far.
>
> If anyone has knowledge of a crosslinker with the above properties I would be thrilled to know about it.
>
> Thanks
>
> Peter Henriksen
>
>
>
>
>
>
> ------------------------------
>
> Message: 4
> Date: Wed, 21 Nov 2007 13:16:51 -0600
> From: "Deitiker, Philip R" <pdeitik from bcm.tmc.edu>
> Subject: RE: Protein/protein-crosslinkers
> To: <Methods from magpie.bio.indiana.edu>
> Message-ID:
>        <B6E27B2A54DE2A419999AF1995EBE21939A24A from BCMEVS6.ad.bcm.edu>
> Content-Type: text/plain;       charset="us-ascii"
>
>
>
> The anhydride bond is one of the most difficult to form.
>
> In peptide synthesis the anhydride that is used to react with the amino
> terminal of the growing peptide is formed by carbodiimide. The anhydride of
> carbonyl groups is unstable and will be attacked by the lysine amino groups causing the permanent formation R-CO-N-R bond.
>
> Typically dicyclohexyl carbodiimide or diisopropyl carbodiimide.
> There are water soluble carbodiimied such as
> 1-ethyl-3-(3-dimethylaminopropyl)carbodiimidic hydrochloride.
> Pierce cat.# 22980. More crossreactivity can be achieved by making a succinic
> anhydride of the protein of interest which has a higher crosslinking activity
> to targets. The target sites are Lysines, amino termini.
>
> This is not suitable for treatment of living cells. Carbodiimides are known as sensitizing agents, they do crosslink proteins on the skin of laboratory researches which can link gliadin (such as used to be used as a donning agent) latex and self proteins. Individuals develop dermatitis to the new
> antigens and this then can cause allergies to gloves, donning agents, etc.
>
> = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = =
>
> There is an enzyme in living cells known as transglutaminase (tTG),
> it can be found in the nucleus, cytosol, and extracellular matrix.
>
> http://en.wikipedia.org/wiki/Tissue_transglutaminase
> http://en.wikipedia.org/wiki/Keratinocyte_transglutaminase
>
> tTG crosslinks 4 amino acid motives (for a discussion of targets
> review literature on gliadin as these have been the most characterized)
> It crosslinks the lysine amino group to glutamines and to a lessor degree apsartamine.
> Guinea pig transglutaminase is available, to be used the target needs to have a 4 amino acid motif that is identified by the enzyme and the enzyme will cross link other proteins to it. This will likely cause ubiquitization and targeting for degradation.
>
> -----Original Message-----
> From: methods-bounces from oat.bio.indiana.edu [mailto:methods-bounces from oat.bio.indiana.edu] On Behalf Of Peter Henriksen
> Sent: Wednesday, November 21, 2007 9:41 AM
> To: methods from magpie.bio.indiana.edu
> Subject: Protein/protein-crosslinkers
>
> I am trying to design an experiment in which I will need to make protein-protein crosslinks but not DNA-protein crosslinks in living cells.
>
> In principal I think this should be possible using a crosslinker that will crosslink between two carboxylic groups but I haven't been able to find such a crosslinker so far.
>
>
> If anyone has knowledge of a crosslinker with the above properties I would be thrilled to know about it.
>
>
>
>
>
> ------------------------------
>
> Message: 5
> Date: Thu, 22 Nov 2007 00:38:14 +0530
> From: "Sudheendra Rao N R" <sudhee26 from gmail.com>
> Subject: Re: troubleshooting + ligation
> To: "Anajli Thomas" <anjalirthomas from gmail.com>,
>        Methods from magpie.bio.indiana.edu
> Message-ID:
>        <a1a1abbb0711211108r799957a3pcf0d479383acef4c from mail.gmail.com>
> Content-Type: text/plain; charset=ISO-8859-1
>
> hi,
> are u using any ligation kit?
> whats the temperature of ligation and whats the time?
> first thing to see is whether its ligated or not
> only then you should proceed towards transformation.
> try overnight ligation at 14 degrees and then..run an agarose gel to see
> your product.
> if its ligated it will become circular and its migration will be faster (u
> can see supercoiled and stuff like that)
> subject that again to single digestion..it will become linear and will match
> with new mol weight (vector+1.5kb)
> then try cloning it.
>
> Sudheendra.
>
> On Nov 21, 2007 10:51 AM, Anajli Thomas <anjalirthomas from gmail.com> wrote:
>
> >      I have been trying to clone a 1.5 kb insert to pEGFP c1 vector. The
> > restriction enzymes used were EcoR1 and Kpn1. I have tried ligation thrice
> > and still no colonies. JM109 was used for transformation.
> >
> > The restriction enzymes were added together for cutting the insert from T
> > vector (primary clone). For ligation I added 5 ul of insert and 2ul of
> > backbone.
> >
> > Could you help me out with this problem
> >
> > Sincerely
> > Anjali
> > _______________________________________________
> > Methods mailing list
> > Methods from net.bio.net
> > http://www.bio.net/biomail/listinfo/methods
> >
>
>
>
> --
> Think before agree
> Think before you nod
> but STOP thinking
> and You Are God
>
>
> ------------------------------
>
> Message: 6
> Date: Thu, 22 Nov 2007 00:45:38 +0530
> From: "Sudheendra Rao N R" <sudhee26 from gmail.com>
> Subject: Re: Protein/protein-crosslinkers
> To: "Peter Henriksen" <peterh from my.molbio.ku.dk>,
>        methods from magpie.bio.indiana.edu
> Message-ID:
>        <a1a1abbb0711211115n2da16be2ref0ae12df336c93 from mail.gmail.com>
> Content-Type: text/plain; charset=ISO-8859-1
>
> hi,
> try these link
> http://www.protocol-online.org/biology-forums/posts/9764.html
>
> http://www.piercenet.com/Products/Browse.cfm?fldID=CE4D6C5C-5946-4814-9904-C46E01232683
>
> https://catalog.invitrogen.com/index.cfm?fuseaction=viewCatalog.viewProductDetails&sku=&productDescription=8259&ref=http%3A%2F%2Fwww%2Egoogle%2Ecom%2Fsearch%3Fq%3Dprotein%2Bprotein%2Bcrosslinker%26rls%3Dcom%2Emicrosoft%3A%2A%26ie%3DUTF%2D8%26oe%3DUTF%2D8%26startIndex%3D%26startPage%3D1
>
>
> I hope its of help to you
>
> Sudheendra.
> On Nov 21, 2007 9:10 PM, Peter Henriksen <peterh from my.molbio.ku.dk> wrote:
>
> > I am trying to design an experiment in which I will need to make
> > protein-protein crosslinks but not DNA-protein crosslinks in living cells.
> >
> > In principal I think this should be possible using a crosslinker that will
> > crosslink between two carboxylic groups but I haven't been able to find such
> > a crosslinker so far.
> >
> > If anyone has knowledge of a crosslinker with the above properties I would
> > be thrilled to know about it.
> >
> > Thanks
> >
> > Peter Henriksen
> >
> >
> >
> >
> > _______________________________________________
> > Methods mailing list
> > Methods from net.bio.net
> > http://www.bio.net/biomail/listinfo/methods
> >
>
>
>
> --
> Think before agree
> Think before you nod
> but STOP thinking
> and You Are God
>
>
> ------------------------------
>
> Message: 7
> Date: Wed, 21 Nov 2007 21:56:37 -0000
> From: "Jose de las Heras" <josenet from tiscali.co.uk>
> Subject: Re: troubleshooting + ligation
> To: methods from net.bio.net
> Message-ID: <5qjo6vFvo65iU1 from mid.individual.net>
>
>
> "Anajli Thomas" <anjalirthomas from gmail.com> wrote in message
> news:mailman.696.1195669227.23109.methods from net.bio.net...
> >      I have been trying to clone a 1.5 kb insert to pEGFP c1 vector. The
> > restriction enzymes used were EcoR1 and Kpn1. I have tried ligation thrice
> > and still no colonies. JM109 was used for transformation.
> >
> > The restriction enzymes were added together for cutting the insert from T
> > vector (primary clone). For ligation I added 5 ul of insert and 2ul of
> > backbone.
> >
> > Could you help me out with this problem
> >
> > Sincerely
> > Anjali
>
> If somebody asked you this, do you think you have all the necessary
> information to be able to suggest anything helpful?
>
> You say nothing of how you purified the fragments, how teh digests looked...
> but what really gets to me is the "For ligation I added 5 ul of insert and
> 2ul of backbone". Even if I know the size of pEGFP, talking microlitres is
> pointless as I don't know teh concentration.
>
> It's one pet hate of mine, when people write protocols etc talking of
> "microlitres" as if we all knew magically what concentration of reagent
> they're using. It makes me think that they don't know. It makes me think
> that they don't really know what is important.
>
> Sorry to sound a bit harsh... but I feel it's got to be said.
>
> Just think about the problem a little bit before you post, so that you're
> able to give the relevant information, and I am sure you will receive more
> helpful answers.
>
> Regards,
>
> Jose de las Heras
>
>
>
>
> ------------------------------
>
> Message: 8
> Date: Thu, 22 Nov 2007 14:18:06 +0800
> From: "WANG XF" <wxfbmn from 163.com>
> Subject: cccDNA preparation from transfected HepG2 cells
> To: methods from magpie.bio.indiana.edu
> Message-ID:
>        <404daa2f0711212218h4af18d7ew3593be5e8a73c148 from mail.gmail.com>
> Content-Type: text/plain; charset=ISO-8859-1
>
> Hi, every one:
>
> Does anyone have a detailed protocol for preparing cccDNA from HepG2 cells
> transfected with HBV supergenome? I have tried the method of Summers, and
> did not get a good result.
>
> Summers method:
> http://jvi.asm.org/cgi/content/abstract/64/6/2819
>
> Thank you all!
>
> LAO Wang
>
>
> ------------------------------
>
> _______________________________________________
> Methods mailing list
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> http://www.bio.net/biomail/listinfo/methods
>
> End of Methods Digest, Vol 30, Issue 14
> ***************************************
>


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



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