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Which buffer is best for DNA Electrophoresis and which is best for Protein to be have a sharp bond?

Considering a higher electrical conductivity compared to TAE & TBE and the generation of less heat. If anyone has a particular formulation (add other materials to improve quality) which is obtained as a result of practical experience. I would be grateful if you send me that.

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    $\begingroup$ The standard for DNA agarose gel is TAE and for the protein, it depends on the size of the protein and the gel type used! Some times MOPS works best and sometimes Tris-acetate works best. It really depends on the gel used and also the protein and its size. You just have to experiment with them and see which works best for you! This document by Invitrogen might be of help (tools.lifetechnologies.com/Content/SFS/ProductNotes/…). To prevent apparatus over heating, put them in a cold room. I can give you my TAE components if you wish! $\endgroup$ – Bez Sep 13 '14 at 12:13
  • $\begingroup$ @Bez please introduse why TAE stand a standard buffer for DNA (are it works for any size of that even in PFGE?).If you have a table for size of proteins and relative buffer that can complete your upper idea about proteins buffer difference please put complete of them in a answer form. $\endgroup$ – M007 Sep 14 '14 at 8:23
  • $\begingroup$ TAE is the standard DNA agarose gel buffer according to Sambrook et al., 1989 and is used by practically every scientist that I know of for running agarose gels for DNA. Please look at figure 2 for the best gel to use for your protein of interest (lifetechnologies.com/uk/en/home/life-science/…). Since the question is on hold I cannot add a new answer but as soon as it opens I will put my comments as a full response. $\endgroup$ – Bez Sep 14 '14 at 9:43
  • $\begingroup$ It would be great if this could be reopened so we can write proper answers and not have to use the comments for this. $\endgroup$ – Chris Sep 14 '14 at 13:37
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The question which buffer for DNA is better is quite old. Both have their pros and cons and I list a few of them:

  • TBE is a better conductor and is thus less prone for overheating the gel
  • Borate is a powerful enzyme inhibitor, so if you want to apply enzymatic steps downstream, TAE is the better choice
  • TAE gives a better resolution for large fragments
  • TBE has a higher resolution for fragments <2kb so it is better here

Both buffers appeared in the 70s when the electrophoresis of nucleic acids was developed (in fact it was derived from protein electrophoresis) and haven't been changed or optimized much since then, although there is a number of disadvantages: First EDTA is not really necessary for DNA electrophoresis (it is for RNA, which is also the most likely reason it is still in there). Then Tris is not the best buffering substance here. Besides being relative expensive it creates a temperature-current feedback loop, meaning the higher the current gets, the higher the temperature will be. This leads to a higher gel temperature with all the negative effects of it. For more details on this, have a look into reference 1.

There has been some research on this matter and there are some suggestions available for better running buffers (details are in references 2 and 3). In short these are:

  • 10mM sodium boric acid (Na2B4O7/Borax): separation of DNA fragments from 100bp-5kbp
  • 5mM lithium acetate (LiOOCCH3): separation of fragments longer than 3 kbp
  • 1mM lithium boric acid (Li2B4O7): separation of small DNA fragments and ssDNA

The borate in this buffers is still problematic in terms of a possible enzymatic inhibition, but this can be overcome with a simple ethanol precipitation of the DNA (which of course also applies for use of TBE buffer).

  1. History and principles of conductive media for standard DNA electrophoresis.
  2. Sodium boric acid: a Tris-free, cooler conductive medium for DNA electrophoresis
  3. Ultra-fast high-resolution agarose electrophoresis of DNA and RNA using low-molarity conductive media
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I have had good experience using a lithium boric acid buffer from Faster Better Media. I use it for RNA gels, but it's advertised for DNA gels. I don't think it can do protein, but I've never tried it. I'm not an electrician, but higher conductivity may be the opposite of what you want. The lithium boric acid buffer claims to have less conductivity than a tris based buffer, allowing you turn the voltage up much higher while producing less heat. The higher voltage pushes the DNA down the gel faster, and keeps the bands sharper. My RNA gels take 30 minutes to run, used to take 3 hours. Of course, those slow gels used a MOPS based buffer and formaldehyde to denature the RNA.

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  • $\begingroup$ The funny thing is one of the founder of Faster Better Media wrote the articles I cited :-) $\endgroup$ – Chris Sep 15 '14 at 12:51
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Grossly, it does not matter what buffer you use. It is the pH that matters.

For DNA electrophoresis EDTA is added in order to chelate divalent cations that serve as cofactors for nucleases. Tris is the base of the buffer and is used to set pH. Along with Tris one can use Boric acid, Acetic acid or phosphoric acid for adjusting the pH.

The buffering range can be obtained by the Henderson-Hasselbalch equation.

The choice of buffers depends on pH range, ease of preparation/storage and fine resolving properties and runtime properties such as heating etc (also mentioned by others).

MOPS is another buffering agent. I am not very sure why MOPS is used instead of Tris for RNA gels; perhaps the latter forms adducts with formaldehyde by Mannich's reaction whereas MOPS cannot undergo that reaction. MOPS is also a Zwitter-ionic species and maintains neutral pH. RNA should not be kept in alkaline pH. At acidic pH, RNA will become neutral and will not move.

For protein electrophoresis things are a little different. Glycine-Chloride buffer is used because it allows stacking.

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There are already many great answers to your question, however I thought I put my comments in form of an answer.

The standard for DNA agarose gel is TAE and for the protein, it depends on the size of the protein and the gel type used! Some times MOPS works best and sometimes Tris-acetate works best. It really depends on the gel used and also the protein and its size. You just have to experiment with them and see which works best for you! This document by Invitrogen might be of help. To prevent apparatus over heating, put them in a cold room. I can give you my TAE components if you wish!

TAE is the standard DNA agarose gel buffer according to Sambrook et al., 1989 and is used by practically every scientist that I know of for running agarose gels for DNA. Please look at figure 2 for the best gel to use for your protein of interest, which has been shown in form of a table.

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