Buffer for Agarose Gel Electrophoresis
Composition of 1x TAE buffer
■ 40 mM Tris (pH 7.6)
■ 20 mM acetic acid
■ 1 mM EDTA
Preparation of 50x TEA stock solution
To prepare 1 liter of 50× TAE dissolve following components in 600 ml of deionized water:
■ 242 g Tris base (FW = 121)
■ 57.1 ml glacial acetic aci
■ 100 ml 0.5 M EDTA (pH 8.0)
Composition of 1x TBE buffer
■ 89 mM Tris (pH 7.6)
■ 89 mM boric acid
■ 2 mM EDTA
Preparation of 10x TBE stock solution
To prepare 1 liter of 10× TBE dissolve following components in
600 ml deionized water:
■ 108 g Tris base (FW = 121)
■ 55 g boric acid (FW = 61.8)
■ 40 ml 0.5 M EDTA (pH 8.0)
Adjust final volume to 1 liter with deionized water.
Use 0.5 X TAE or TBE for electrophoresis
Standard 1 x TBE buffer produces sharp bands and good resolution in gels run at typical low voltages of ~ 10 V/cm. However, such gels usually require run times of 50 – 90 min to achieve optimum separation of DNA fragments
TBE: good resolution
Use of 0.5 x TB also produced good separation of bands at voltages up to 250 V, though resolution was compromised at 300 V
Gels prepared and run in 0.3 x TB exhibited low resolution and compression of bands toward each other at all voltages, with strongest compression and non-uniform band intensities apparent at 250 and 300 V. Thus, good resolution was achieved using both 0.5 x TB and 1 x TB at voltages up to 250 V (25 V per cm), though all bands were closer to each other at the higher voltages. EDTA is not essential for electrophoresis and is added as a preventative to inhibit DNA nucleases