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Yvonne Couch | 6 Feb 2012 16:31
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Tris-HEPES

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
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Nikola Loncar | 7 Feb 2012 18:26
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Re: Methods Digest, Vol 81, Issue 2

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.

________________________________
 From: "methods-request@..." <methods-request <at> oat.bio.indiana.edu>
To: methods@... 
Sent: Tuesday, February 7, 2012 6:07 PM
Subject: Methods Digest, Vol 81, Issue 2

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Nikola Wenta | 7 Feb 2012 18:33
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Re: Tris-HEPES

> 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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Virash Gupta | 8 Feb 2012 05:35
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Re: Methods Digest, Vol 81, Issue 2-'50mM tris-HEPES buffer (ph7.5)'.

'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)

On Tue, Feb 7, 2012 at 10:37 PM, <methods-request@...>wrote:

> Send Methods mailing list submissions to
>        methods@...
>
> To subscribe or unsubscribe via the World Wide Web, visit
>        http://www.bio.net/biomail/listinfo/methods
> or, via email, send a message with subject or body 'help' to
>        methods-request@...
>
> You can reach the person managing the list at
>        methods-owner@...
>
> When replying, please edit your Subject line so it is more specific
> than "Re: Contents of Methods digest..."
>
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> Today's Topics:
>
>   1. Tris-HEPES (Yvonne Couch)
>   2. Re: Tris-HEPES (DK)
>
>
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Virash Gupta | 8 Feb 2012 18:32
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Re: Methods Digest, Vol 81, Issue 3

'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@...>
>> Subject: Re: Methods Digest, Vol 81, Issue 2
>> To: "methods@..." <methods@...>
>> Message-ID:
>>        <1328635564.90246.YahooMailNeo@...>
>> 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
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Bjorn | 15 Feb 2012 08:45
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Desperately looking for E. coli with lambda cI857 Sam7

Hi all,
I am looking for an E. coli strain with a lysogenic lambda phage that is practical to induce and purify, such
as one with cI857 Sam7.

If you have one, I would be most grateful if you could send it to me.
Alternatively, point me to a resource that is free or not too expensive where I could buy one.

I would like to use it to produce DNA for my own molecular weight markers.
I have found it much harder to find this than I would have thought.

Thanks,

Björn Johansson
Departament of Biology
University of Minho
Campus de Gualtar
4710-057 Braga
PORTUGAL
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Duncan Clark | 15 Feb 2012 16:56

Re: Desperately looking for E. coli with lambda cI857 Sam7

Historians believe that in newspost 
<26192644.3453.1329291940527.JavaMail.geo-discussion-forums <at> vbmh19> on 
Tue, 14 Feb 2012, Bjorn <bjornjobb@...> penned the following 
literary masterpiece:
>I am looking for an E. coli strain with a lysogenic lambda phage that is practical to induce and purify, such
as one with cI857 Sam7.
>
>If you have one, I would be most grateful if you could send it to me.
>Alternatively, point me to a resource that is free or not too expensive where I could buy one.

Buy some standard full length lambda cI857 Sam7 methylated DNA from NEB 
and electroporate into suitable E.coli host. Screen for lysogen i.e. 
grows at 28C but not 42C.

Or buy in same DNA but find someone with a lambda packing kit, package, 
plate screen etc.

Duncan
--

-- 
I love deadlines. I especially like the whooshing noise they make as
they go flying by.

Duncan Clark
GeneSys Ltd.
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Bjorn | 16 Feb 2012 08:50
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Re: Desperately looking for E. coli with lambda cI857 Sam7

On Feb 15, 3:56 pm, Duncan Clark <blackh...@...> wrote:
> Historians believe that in newspost
> <26192644.3453.1329291940527.JavaMail.geo-discussion-forums <at> vbmh19> on
> Tue, 14 Feb 2012, Bjorn <bjornj...@...> penned the following
> literary masterpiece:
>
> >I am looking for an E. coli strain with a lysogenic lambda phage that is practical to induce and purify,
such as one with cI857 Sam7.
>
> >If you have one, I would be most grateful if you could send it to me.
> >Alternatively, point me to a resource that is free or not too expensive where I could buy one.
>
> Buy some standard full length lambda cI857 Sam7 methylated DNA from NEB
> and electroporate into suitable E.coli host. Screen for lysogen i.e.
> grows at 28C but not 42C.
>
> Or buy in same DNA but find someone with a lambda packing kit, package,
> plate screen etc.
>
> Duncan
> --
> I love deadlines. I especially like the whooshing noise they make as
> they go flying by.
>
> Duncan Clark
> GeneSys Ltd.

Thanks a lot! but how do I select for transformants? does it have a
selection marker?
sorry, never worked with lambda...
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Nadeem Ahamad | 16 Feb 2012 10:46
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Can I have PDF of LPS extraction method described by Westphal, 1965 ???

--

-- 
*Nadeem,*
*Ph.D. **Scholar*
*Tissue Engineering and Drug delivery Lab *
*Biological Science And **Bioengineering*
*Indian Institute Of Technology Kanpur ( I.I.T.K),*
*Kanpur-208016 , India*
*Mob - +919793980077*
*
*
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Gmane