Preparation of TAE Buffer..
Madhura Sagare
Friday, 21 November 2008 09:28 UTC
Hello Everyone,
Can we use Tris-HCl instead of Tris base in the preparation of TAE buffer?
Can the other components i.e. Glacial Acetic Acid and EDTA be kept unchanged?
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Replies
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No… Tris-HCl is, by definition, buffered. You want Tris base so that it’s buffered with the acetic acid.
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Ok..
So what is the difference(in buffering potential in using
TAE buffer with Tris base..
and
TAE buffer with Tris-HCl? -
Never tried this, but here is what I would reason to be the case: As long as the final pH, and concentration of Tris and Acetate ions are the same, buffering power should be ~the same. However, the addition Cl- ions will be there… and the pH using Tris Cl will probably be lower than expected in the final TAE (as compared to Tris base) and require some NaOH to pH to 8. Therefore I would expect additional Na+ and Cl- ions in a TAE prepared using Tris Cl. These may have a sufficient effect on the conductivity of the TAE as to change the expected buffer behavior in electrophoresis. Beyond this, I would expect no major differences.
..someone correct me if I am wrong, as I would appreciate to know any factor I may have failed to consider.
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John is right. The pH will be wrong, and the ionic strength will be wrong.
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Exactly, I agree with John’s explanation that..
The conductivity of the TAE Buffer would be changed.
I doubt, whether such a type of buffer will be ok for carrying out electrophoresis of DNA. -
Madhura,
I agree with Richard and John, don’t substitute Tris-HCl for Tris base when preparing TAE buffer. TAE was specifically developed for use in electrophoresis and the Cl ion will change the conductivity of the buffer. I can tell you this from past experience because an undergraduate intern working for us mistakenly prepared a batch of TAE with Tris-HCl and then ended up melting the gel and frying a power supply when she tried to run the electrophoresis.
Sheldon
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the pH Tris HCl in water will be around 5.0 where as the Tris Base pH will be ~11.0. In TAE or TBE acetic acid or boric acid will be used to bring Tris pH to desired pH to use it for electrophoresis. If you use Tris Hcl and an acid (Acetic or boric) the resultant pH of the buffer will be acidic in nature. In an electrophoresis system DNA need to move towards positive side. At acidic pH the DNA will not have negative charge to move towards positive side. To prepare TAE or TBE fixed amount of Tris base and acid are used to get desired pH. The charge of the molecules depends on the pH (it should be more than its pka to have negative charge. If you want to use Tris HCL for preparation of these buffers probably you need to increase the Tris HCl solution pH to ~11.0 using NaOH then add required amount of boric or acidic acid ( I never tried it, I guess it may work!)
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Thanks Sheldon, for sharing the hazardous consequences!!
Pandurang,
Even I thought of adjusting the pH by using NaOH.But,wouldn’t the excess Na ions cause precipitation of DNA and hinder its mobility in the gel?
So, I avoided the preparation of this messy buffer and finally I preferred making the TAE buffer using the Tris-Base and not Tris-HCl.
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