Methods Digest, Vol 81, Issue 3

Virash Gupta via methods%40net.bio.net (by virashkgupta from gmail.com)
Wed Feb 8 12:32:19 EST 2012


'50mM tris-HEPES buffer (ph7.5)'.
Suspend 6.055  g tris base in one litre MQ water and adjust pH to 7.5 by
adding solid HEPES by continuousely mixing by keeping the container on a
magnetic stirrir. This will take quite a time to become stabilized. HEPES
is an acidic component and will substitute HCL used for making Tris CL (to
neutralize Tris base)


>>
>> ----------------------------------------------------------------------
>>
>> Message: 1
>> Date: Tue, 7 Feb 2012 09:26:04 -0800 (PST)
>> From: Nikola Loncar <hemicar085 from yahoo.com>
>> Subject: Re: Methods Digest, Vol 81, Issue 2
>> To: "methods from oat.bio.indiana.edu" <methods from oat.bio.indiana.edu>
>> Message-ID:
>>        <1328635564.90246.YahooMailNeo from web111708.mail.gq1.yahoo.com>
>> Content-Type: text/plain; charset=iso-8859-1
>>
>> Hi there,
>>
>>
>> I disagree with DK since molarity of buffer can not be calculated as a
>> sum of two component. Lets take MES acetate as an example. 1 M buffer means
>> that there is one mole of MES in 1 L of buffer whose pH is adjusted with
>> acetic acid.
>>
>> Concerning tris hepes buffer - is it possible that your colleague meant
>> that you can use either tris or hepes? otherwise, you can dissolve hepes to
>> be 50 mM and adjust pH with concentrate solution of tris (tris solution is
>> by itself very basic - pH ~11), or other way around but i can not remember
>> any explanations why anyone would use that buffer.
>>
>>
>> best wishes,
>> N.L.
>>
>>
>>
>> ________________________________
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>> To: methods from magpie.bio.indiana.edu
>> Sent: Tuesday, February 7, 2012 6:07 PM
>> Subject: Methods Digest, Vol 81, Issue 2
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>> Today's Topics:
>>
>>    1. Tris-HEPES (Yvonne Couch)
>>    2. Re: Tris-HEPES (DK)
>>
>>
>> ----------------------------------------------------------------------
>>
>> Message: 1
>> Date: Mon, 6 Feb 2012 15:31:15 +0000
>> From: Yvonne Couch <yvonne.couch from pharm.ox.ac.uk>
>> Subject: Tris-HEPES
>> To: "methods from magpie.bio.indiana.edu" <methods from magpie.bio.indiana.edu>
>> Message-ID:
>>     <FC9D052C4A50C04E8D6C54D2F870A5819380041B8F from EXMBX01.ad.oak.ox.ac.uk>
>> Content-Type: text/plain; charset="us-ascii"
>>
>> Hi all,
>> I am trying to replicate the experiments of an ex-colleagues whose notes
>> require '50mM tris-HEPES buffer (ph7.5)'. I am not sure whether the 50mM
>> refers to the tris or the HEPES and was wondering whether anyone had any
>> experience with this? I am using it as an extraction buffer for fresh
>> tissue (the other components of which are sucrose and EDTA) and need to
>> maintain the integrity of the enzymes within the tissue, rather than the
>> cells. If anyone has any suggestions for a replacement for this buffer or
>> any more details on how to make a tris-HEPES buffer, I would be most
>> grateful.
>> Regards
>> Yvonne
>>
>>
>>
>> ------------------------------
>>
>> Message: 2
>> Date: Tue, 07 Feb 2012 02:18:25 GMT
>> From: dk from no.email.thankstospam.net (DK)
>> Subject: Re: Tris-HEPES
>> To: methods from net.bio.net
>> Message-ID: <QR%Xq.9509$rV2.1695 from newsfe11.iad>
>>
>> In article <mailman.222.1328552955.3721.methods from net.bio.net>, Yvonne
>> Couch <yvonne.couch from pharm.ox.ac.uk> wrote:
>> >Hi all,
>> >I am trying to replicate the experiments of an ex-colleagues whose notes
>> > require '50mM tris-HEPES buffer (ph7.5)'. I am not sure whether the 50mM
>> > refers to the tris or the HEPES and was wondering whether anyone had any
>> > experience with this?
>>
>> There is plenty of ambiguity there but I'd think that it means that
>> the buffer is made with 25 mM of Tris and 25 mM of HEPES.
>> At least that's how it works in our lab: a "1.0 M MES-Acetate"
>> is 0.5 M of each component.
>>
>> > has any suggestions for a replacement for this buffer
>>
>> It's going to be a very good buffer at 7.5. Lots of things could
>> work as replacement though.
>>
>> >or any more details on
>> > how to make a tris-HEPES buffer, I would be most grateful.
>>
>> Mix HEPES (free acid form) and Tris (free base form) and
>> adjust pH to 7.5 with either NaOH or HCl (I am not sure what
>> pH is going to be when mixing equal concentrations of
>> HEPES and Tris).
>>
>> DK
>>
>>
>>
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>> ------------------------------
>>
>> Message: 2
>> Date: Tue, 7 Feb 2012 17:33:12 +0000
>> From: Nikola Wenta <Nikola.Wenta from nottingham.ac.uk>
>> Subject: Re: Tris-HEPES
>> To: "methods from net.bio.net" <methods from magpie.bio.indiana.edu>
>> Message-ID:
>>        <
>> 5D45216BCE0CB845A9E43697AE5ED1E72D5A19C733 from EXCHANGE2.ad.nottingham.ac.uk>
>>
>> Content-Type: text/plain; charset="us-ascii"
>>
>> > Hi all,
>> > I am trying to replicate the experiments of an ex-colleagues whose
>> > notes require '50mM tris-HEPES buffer (ph7.5)'. I am not sure whether
>> > the 50mM refers to the tris or the HEPES and was wondering whether
>>
>> I guess it is achieved by titrating a 50 mM unbuffered TRIS solution
>> against a 50 mM unbuffered HEPES solution (or the other way around) until
>> pH is 7.5. The molarity actually only corresponds to the buffer capacity of
>> the buffer, so the initial molarity of the "stock" buffer shouldn't matter
>> as long as the effective final concentration of the buffer is not too low,
>> i.e. the buffer has lost its buffer capacity. This implies that the
>> colleague initially actually prepared a 500 mM TRIS-HEPES buffer with pH
>> 7.5.
>> Just a thought: usually pH of TRIS would be adjusted with acid (HCl),
>> while for HEPES a base (NaOH) would be used.
>> Cheers,
>> Niko
>>
>> p.s.:
>> > There is plenty of ambiguity there but I'd think that it means that
>> > the buffer is made with 25 mM of Tris and 25 mM of HEPES.
>> > At least that's how it works in our lab: a "1.0 M MES-Acetate"
>> > is 0.5 M of each component.
>>
>> I don't agree with DK's statement. Mixing lower molarities of buffers
>> usually yields even lower molarities == dilution.
>>
>>
>>
>>
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>
>
>
> --
> Dr V K Gupta
> Sr Microbiologist (Mol Biology)
> IMBL, Department of Entomology
> Pun. Agric. Univ., Ludhiana (Pb)-141004- India
> M: 081465-55515
>



-- 
Dr V K Gupta
Sr Microbiologist (Mol Biology)
IMBL, Department of Entomology
Pun. Agric. Univ., Ludhiana (Pb)-141004- India
M: 081465-55515


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