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Appropriate Buffer for electerophoresis of DNA & Protein TBE or TAE?
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Albert Addae
Appropriate Buffer for electerophoresis of DNA & Protein TBE or TAE?
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.
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.
The funny thing is one of the founder of Faster Better Media wrote the articles I cited :-)More
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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.
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.
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).
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 thegel
Borate is a powerful enzyme inhibitor, so if you want to applyenzymatic 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). ThenTris 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 thenegative 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 fragmentsfrom 100bp-5kbp
5mM lithium acetate (LiOOCCH3): separation of fragments longer than 3kbp
1mM lithium boric acid (Li2B4O7): separation of small DNA fragmentsand ssDNA
The borate in this buffers is still problematic in terms of a possible enzymatic inhibition, but this can be overcome with a simple ethanolprecipitation of the DNA (which of course also applies for use of TBE buffer).
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.
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.
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.
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.
More
VOTE
VOTE
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.
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.
More
VOTE
The question which buffer for DNA is better is quite old. Both have their pros and cons and I list a few of them:
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:
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).
The question which buffer for DNA is better is quite old. Both have their pros and cons and I list a few of them:
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). ThenTris 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 thenegative 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:
The borate in this buffers is still problematic in terms of a possible enzymatic inhibition, but this can be overcome with a simple ethanolprecipitation of the DNA (which of course also applies for use of TBE buffer).
More
VOTE
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.
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.
More
VOTE