Methods Digest, Vol 19, Issue 6

[Virash Gupta]

To Cristiano Fontes,
I will not think Silver will compete with Mg in PCR reaction. Simple reason
every enzyme has its specific cofactors. Taq may not work with Ag as it
needs Mg. In case of doubt I suggest to run a simple PCR reaction using
AgCl2, MgCl2  and combination of both at 1.5mM conc and check the
amplification differences. That can help you satisfy practically. If Ag does
not interfere, you can straight use cut bands from silver stained gels for
PCR amplification

V K Gupta


On 12/7/06, methods-request At oat.bio.indiana.edu <
methods-request At oat.bio.indiana.edu> wrote:
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> Today's Topics:
>
>   1. RE: sequence apparatus (brenda hendriks)
>   2. Re: Plasmid as a template PCR (Pow Joshi)
>   3. I am looking for a protocol to purify DNA drom silver stained
>      PAGE      Gels (Cristiano Fontes)
>   4. EtBr decontamination (Pow Joshi)
>   5. Proteins (camelia vlad)
>   6. Re: EtBr decontamination (Han)
>
>
> ----------------------------------------------------------------------
>
> Message: 1
> Date: Wed, 6 Dec 2006 15:26:58 +0000
> From: brenda hendriks <b_hendriks_stegeman At hotmail.com>
> Subject: RE: sequence apparatus
> To: <methods At magpie.bio.indiana.edu>
> Message-ID: <BAY110-W47656A7A11773D047C29FABDD0 At phx.gbl>
> Content-Type: text/plain; charset="iso-8859-1"
>
> Hi there,For education of our students we are looking for a ABI sequencer
> in the Netherlands. We would be very pleased having a sequncer where the
> students have to make their own gels.I hope there is someone who can help
> me out here.All the best,Brenda
> _________________________________________________________________
> Probeer Live.com: je eigen persoonlijke opstartpagina met alleen de dingen
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> ------------------------------
>
> Message: 2
> Date: Wed, 6 Dec 2006 11:15:52 -0500
> From: "Pow Joshi" <pow.joshi At gmail.com>
> Subject: Re: Plasmid as a template PCR
> To: ati405 At gmail.com
> Cc: Methods At magpie.bio.indiana.edu
> Message-ID:
>        <710764ea0612060815q2096fe9u2170c47ac3939d5e At mail.gmail.com>
> Content-Type: text/plain; charset=ISO-8859-1; format=flowed
>
> On 6 Dec 2006 02:57:19 -0800, ati405 At gmail.com <ati405 At gmail.com> wrote:
> > Hi
> > I cloned one beta-thalassemia mutation (Homo) in t-vector
> > but when I do ARMS-PCR ....I see 2 band...one for mutant and another
> > for noraml !!!!!
> > I have to see only one band for mutant gene!!
> > I change several factors...but it reapeat....
> > plaese help me...
> > It's very important!!!
>
>
> you may be having some contamination problem.... could you give more
> details about the where you isolate the plasmid from, etc.,
> pow
> > _______________________________________________
> > Methods mailing list
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>
>
>
> ------------------------------
>
> Message: 3
> Date: Wed, 6 Dec 2006 17:08:49 -0200
> From: "Cristiano Fontes" <cristiano.fontes At canavialis.com.br>
> Subject: I am looking for a protocol to purify DNA drom silver stained
>        PAGE    Gels
> To: <methods At magpie.bio.indiana.edu>
> Message-ID: <20061206190826.E233D7C60F At ns.canavialis.com.br>
> Content-Type: text/plain;       charset="us-ascii"
>
> Hello, i am doing my own sizestandart so I can make it with the exact band
> sizes I have and I would like to find a way to purify the DNA I run in a
> 8%
> polyacrilamid Gel, colored with silver. The thing is that I am going to
> use
> this DNA into a PCR reaction and I think silver will compete with
> magnesium
> In the reaction giving me a bad reaction and low DNA.
>
> Is there any nice protocol to extract the DNA from the Gel and get rid of
> the Silver ? or the silver doesn't compete with Mg ?
>
> Thanks
>
> Cristiano Fontes
>
>
> ------------------------------
>
> Message: 4
> Date: Wed, 6 Dec 2006 18:26:31 -0500
> From: "Pow Joshi" <pow.joshi At gmail.com>
> Subject: EtBr decontamination
> To: Methods At magpie.bio.indiana.edu
> Message-ID:
>        <710764ea0612061526k14988aaet7e47b3ea682d5221 At mail.gmail.com>
> Content-Type: text/plain; charset=ISO-8859-1; format=flowed
>
> Hi everyone,
>
> I was wondering if anyone would know the details of decontaminating
> EtBr;  and if it interacts with Bleach to form any kind of toxic
> intermediate or product.
>
> thanks
> pow
>
>
>
> ------------------------------
>
> Message: 5
> Date: Thu, 7 Dec 2006 04:10:13 -0800 (PST)
> From: camelia vlad <camivlad At yahoo.com>
> Subject: Proteins
> To: methods At magpie.bio.indiana.edu
> Message-ID: <981979.83760.qm At web39612.mail.mud.yahoo.com>
> Content-Type: text/plain; charset=iso-8859-1
>
> Hi,
>
> Please send me the protocol for proteins intact extraction from gel(
> camelia.vlad At uni-konstanz.de).I have runed a SDS-PAGE with an
> antibody(150KDa) and I want to recover it and to run another gel after
> denaturation and reduction.
>
>
> Thanks a lot!,
>
> Camelia Vlad
>
>
> ---------------------------------
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> ------------------------------
>
> Message: 6
> Date: Thu, 07 Dec 2006 10:57:28 GMT
> From: Han <nobody At nospam.not>
> Subject: Re: EtBr decontamination
> To: methods At net.bio.net
> Message-ID: <Xns98923CB822BAEikkezelf At 199.45.49.11>
>
> "Pow Joshi" <pow.joshi At gmail.com> wrote in
> news:mailman.318.1165454886.19683.methods At net.bio.net:
>
> > Hi everyone,
> >
> > I was wondering if anyone would know the details of decontaminating
> > EtBr;  and if it interacts with Bleach to form any kind of toxic
> > intermediate or product.
> >
> > thanks
> > pow
>
> Google is your friend.  One refernce found with a search for "clean up
> ethidium bromide" is:
> <http://info.anu.edu.au/hr/OHS/Hazard_Alerts/_Ethidium_Bromide.asp>
>
> HTH
> --
> Best regards
> Han
> email address is invalid
>
>
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> End of Methods Digest, Vol 19, Issue 6
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>