'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 <[email protected]> >> Subject: Re: Methods Digest, Vol 81, Issue 2 >> To: "[email protected]" <[email protected]> >> Message-ID: >> <[email protected]> >> 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. >> >> >> >> ________________________________ >> From: "[email protected]" < >> [email protected]> >> To: [email protected] >> Sent: Tuesday, February 7, 2012 6:07 PM >> Subject: Methods Digest, Vol 81, Issue 2 >> >> Send Methods mailing list submissions to >> [email protected] >> >> 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 >> [email protected] >> >> You can reach the person managing the list at >> [email protected] >> >> When replying, please edit your Subject line so it is more specific >> than "Re: Contents of Methods digest..." >> >> >> 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 <[email protected]> >> Subject: Tris-HEPES >> To: "[email protected]" <[email protected]> >> Message-ID: >> <fc9d052c4a50c04e8d6c54d2f870a5819380041...@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: [email protected] (DK) >> Subject: Re: Tris-HEPES >> To: [email protected] >> Message-ID: <QR%[email protected]> >> >> In article <[email protected]>, Yvonne >> Couch <[email protected]> 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 >> >> >> >> ------------------------------ >> >> _______________________________________________ >> Methods mailing list >> [email protected] >> http://www.bio.net/biomail/listinfo/methods >> >> End of Methods Digest, Vol 81, Issue 2 >> ************************************** >> >> ------------------------------ >> >> Message: 2 >> Date: Tue, 7 Feb 2012 17:33:12 +0000 >> From: Nikola Wenta <[email protected]> >> Subject: Re: Tris-HEPES >> To: "[email protected]" <[email protected]> >> Message-ID: >> < >> 5d45216bce0cb845a9e43697ae5ed1e72d5a19c...@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. >> >> >> >> >> This message and any attachment are intended solely for the addressee and >> may contain confidential information. If you have received this message in >> error, please send it back to me, and immediately delete it. Please do >> not use, copy or disclose the information contained in this message or in >> any attachment. Any views or opinions expressed by the author of this >> email do not necessarily reflect the views of the University of Nottingham. >> >> This message has been checked for viruses but the contents of an >> attachment >> may still contain software viruses which could damage your computer >> system: >> you are advised to perform your own checks. Email communications with the >> University of Nottingham may be monitored as permitted by UK legislation. >> >> >> >> ------------------------------ >> >> _______________________________________________ >> Methods mailing list >> [email protected] >> http://www.bio.net/biomail/listinfo/methods >> >> End of Methods Digest, Vol 81, Issue 3 >> ************************************** >> > > > > -- > 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 _______________________________________________ Methods mailing list [email protected] http://www.bio.net/biomail/listinfo/methods
