TBE buffer is recommended for a smaller DNA size fragments (less than 1 kb), it gives sharper bands. TAE is great for large DNA fragment, particularly for more than 10 kb. However, if you are planning to do DNA isolation from the gel, it is better to use TAE buffer to get better DNA yield when using gel extraction kits.
TBE buffer is recommended for a smaller DNA size fragments (less than 1 kb), it gives sharper bands. TAE is great for large DNA fragment, particularly for more than 10 kb. However, if you are planning to do DNA isolation from the gel, it is better to use TAE buffer to get better DNA yield when using gel extraction kits.
We have always worked with TAE buffer although TBE is recommended for smaller DNA size fragments. We have worked with as low as 100 bp DNA with TAE for cloning.
DNA gel electrophoresis may be carried out in TAE or TBE. I have used both and it worked fine for me. As far as I read, Tris-acetate has a low buffering capacity and it tends to become exhausted during extended electrophoresis (the anode becomes alkaline, the cathode becomes acidic) whereas Tris-borate has higher buffering capacity and can be used extensively. Since ion strength of TBE is higher than TAE, solutes in TBE move faster than in TAE. Moreover, temperature of gel increases using TAE-gel for a long time, pH might significantly decrease because of Tris's temperature dependency of pKa. TAE buffer being a weaker buffer, crack more easily than TBE. So when it is necessary to run at high voltages, TBE is more often preferred, but resolution suffers a bit. TAE requires more time to run but provides the better resolution.
I would like to ask Anna Konovalova about the reason behind the fact that why TAE would allow better resolution of larger fragments (because it has lesser buffering capacity and would thus run slow?) and why would it give better gel elution efficiency than TBE? I read the article shared by Andrea Ray and it says about the downstream processes being affected by the borate carry-overs. Should that affect elution efficiency as well?
Tias, there is a phenomenon called electroendosmosis (EEO), resulting in flow of water toward the cathode (against the direction of the DNA migration). It happens because the agarose has a fixed negative charge. In an electric field, immobilized agarose charge groups (primarily sulfate and carboxyl groups) cannot migrate, but their counter (positive) ions do migrate, moving toward the cathode, causing a net flow of water through osmosis in that direction. This opposite flow of water retards the DNA movement but the effect is only noticeable for large DNA fragments which already migrate slowly.
TAE buffer in combination with low field strength (1-2 V/cm) is recommended because it helps to lower EEO during long runs. It improves resolution while decreasing DNA smearing. But since it has lower buffer capacity, for long runs (e.g. 16 hrs) you'd need to replace the buffer periodically.
But then again, it is only a concern for these specialized DNA electrophoresis applications. For basic purposes, using TBE and TAE is a personal preference.
As for DNA extraction, I don't know why, but TBE can interfere with some steps, many kits recommend to use TAE for better yields. In addition, borate is a strong inhibitor of many enzymes, and borate carry over during the DNA extraction can inhibit downstream applications, for example, cloning. Ligase is particularly sensitive. Some protocol recommend using an excess of any enzyme if DNA was purified from TBE agarose gels.
OK, so apparently borate ion form highly stable DNA/borate complexes which interfere with DNA binding to silica-based columns, lowering the final DNA yield after extraction.
Yes, you can use TBE. We use 1x TBE for agarose gel prep for bands up to 2 Kb max and we get clear results.
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