C.B.S. Scientific SGE-040-02 - Manual (Page 11)

C.B.S. œ Scientific Econo-Sub Units 11
The recommended power conditions for optimal resolution are 50 to 150VDC, constant, and 25 to
80 mA. The usual run time will vary for the voltage chosen but should range from 15 to 60
minutes, monitoring nucleic acid migration by progress of marker dyes. Constant power is not a
necessity, but it produces uniform heat throughout the run. 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*
Buffer Volumes:
Cat. # Gel Bed dimensions Liters
SGE-014-02 14cm x 20cm 1.6
SGE-020-02 20cm x 20cm 2.2
SGE-030-02 20cm x 30cm 2.3
SGE-040-02 20cm x 40cm 2.4
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
μg/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.
4.3 References
1. Hames, B.D., Rickwoood, D. (ed.) (1990). Gel Electrophoresis of Nucleic Acids.
A Practical Approach. 2
nd
edn. IRL Press, Oxford. Ch. 2.
2. Sambrook, J., Fritsch, E.F., Maniatis, T. (1989). Molecular Cloning. A Laboratory
Manual. 2
nd
edn. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New
York. Ch 6.
3. Ausubel, F.M., Brent, R., Kingston, R.E., Moore, D.D., Seidman, J.G., Smith, J.A.,
Struhl, K. (ed) (1993). Current Protocols in Molecular Biology. Vol. 1, Greene
Publishing Associates, Inc. and John Wiley & Sons, Inc., Ch. 2.