PREPARATION OF TAE BUFFER

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Notes:

TAE = tris acetate EDTA buffer

TAE Buffer (50X) is a solution used in agarose gel electrophoresis experiments’ and it is typically used for the separation of nucleic acid molecules (i.e., DNA and RNA). It can also be used as a running buffer for other preparative work in molecular biology experiments.

Aside TAE, tris borate EDTA (TBE) is another important buffer used for molecular biology experiments including gel electrophoresis. Both TAE and TBE are the two most common running buffers used in nucleic acid electrophoresis. They have a constant pH and are able to conduct electricity because of their concentration of hydrogen ions = which is why they are the most commonly used buffer in gel electrophoresis and/or molecular biology experiments in the microbiology laboratory.

DNA mobility on agarose gel electrophoresis is known to depend on the composition and strength of electrode buffer as well as the agarose concentrations. The resolution of super coiled DNA is better in TAE than TBE but TAE’s buffering capacity is rather low as it tends to become exhausted during successive electrophoresis. TAE buffer is more useful for larger DNA fragments (<2.0kb) but TBE is more effective to obtain higher resolution of smaller DNA fragments (i.e., 300bp). Either buffer is adequate is applicable for middle-sized DNA fragments.

For the preparation of 1 liter (1000 ml) of 50 x TAE buffer mix:

242 g        Tris free base    

18.61 g     Disodiumn EDTA   

57.1 ml     Glacial Acetic Acid    

Add Tris and EDTA to approximately 700 ml H2O and stir until dissolved. 

Add the acetic acid and adjust the volume to 1 liter.

The pH (around 8.6) is generally not adjusted.

Dilute 1:100 in H2O for 0.5 x running buffer.

For a small 1% agarose gel, add 0.25 g agarose to 25ml of 0.5 x running buffer, boil in microwave and let cool down to ~60°C.

Mix 10µl of 6xTriTrack loading dye with 5µl midori green (stored in fridge);

Load 5µl DNA sample with 1ul of loading dye+midori green mixture and run gel at 100V in the transilluminator.

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