C.B.S. Scientific WSGE-014 - Manual (Page 14)

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SECtIOn 4
Running Conditions
4 1 Recommended Power
As a rule, optimal resolution of larger molecules is achieved during longer runs at lower voltages, whereas smaller
molecules require shorter runs at higher voltages. Applied voltage gradients can therefore be anywhere in the
range of 1-10 VDC/cm of gel. Using the standard buffer systems listed below, most runs will use 5 VDC/cm as a
general rule.
The usual run time will vary for the voltage and pathlength chosen but should range from 15 to 60 minutes. Nucleic
acid migration is monitored by the progress of marker dyes. Constant power is not a necessity, but it produces
uniform heat throughout the run, therefore minimizing band diffusion. Be sure the polarity is correct i.e. that the DNA
is loaded near the cathode (black electrode) to run toward the anode (red terminal).
Agarose gels may be stored for several days at 4°C wrapped in plastic wrap. Seakem Agarose (FMC) is used
(normally) for preparative and analytical gels. Other types of agarose can be used for special purposes.
4 2 Recommended buffers*
Type* Concentrated Stock/liter Final Concentration
TAE 50X - 242 gm Tris base 1X - 0.04M Tris-acetate
(Tris-acetate) 57.1ml glacial acetic acid 0.001M EDTA
100 ml 0.5M EDTA (pH 8.0)
TBE 5X - 54 gm Tris base 0.5X - 0.045M Tris-borate
(Tris-borate) 27.5 gm boric acid 0.001M EDTA
20 ml 0.5M EDTA (pH 8.0)
10X - Loading Buffer (DNA)*
0.25% Bromophenol blue
0.25% Xylene cyanol
20% Ficoll Type 400
0.1M EDTA, pH 8.0
Ethidium Bromide Staining
EtBr can be premixed with buffers and agarose for use during electrophoresis. Add to
agarose only after temperature has fallen below 55°C. Gels can also be stained after
electrophoresis in a soaking tray. Use EtBr at a final concentration of 0.1*g/ml from a
1mg/ml stock solution. Ethidium Bromide is a powerful mutagen. ALWAYS wear
gloves, eye protection and protective clothing. Dispose of solutions in
accordance with the safety regulations of your institution.