The Sequi-GenŽ GT is a vertical slab gel electrophoresis unit used for high resolution nucleic acid gel electrophoresis, which is crucial in experiments like DNase foot-printing, DNA finger-printing, RNase protection assay, primer extension analysis, etc. Bio-Rad claims the Sequi-GenŽ has the most advanced DNA sequencing cell available and indeed it is. Casting long gels is generally difficult and traditional methods are based on glass plate sliding or plate dropping methods, which most of the time involve problems with plate-sealing with tape, bubble formation in the gel and leaking/spilling of acrylamide mix, which is neurotoxic. The best feature of the Sequi-GenŽ GT is the gel-casting which uses a syringe method that allows fast and easy casting.
The Sequi-GenŽ GT includes 1) A universal base: a lower buffer chamber that can accept various gel dimensions (21 x 40/50 cm, 38 x 30/50 cm), 2) An integral plate chamber (IPC) which is a permanently sealed upper buffer chamber that takes care of uniform heat dissipation preventing smiling patterns and allowing leak-free electrophoresis, 3) Full-length lever-operated clamps that easily slide onto the gel sandwich preventing contact with the edges of the glass plates, 4) A long plate made up of chemically tempered glass that can withstand heat (generated during electrophoresis), 5) Top and bottom safety covers that offer electrical protection, and 6) A Stabilizer bar to hold the gel assembly in a vertical orientation. The other feature is a drain port for easy buffer disposal, which is especially helpful in discarding radioactive waste. For gel casting, a caster base, syringe and tubing are provided. Also vinyl spacers and a sharkstooth comb (0.4 mm) are provided.
We study DNA-protein interactions by DNaseI footprinting assays where high resolution separation of nucleotides is desired. For sequencing, generally 6-8% urea poly-acrylamide gels are employed. For better gel removal, we always siliconize the long glass plate with SIGMACOTEŽ
and wipe the IPC plate with NaOH solution to make its surface rough, so that when plates are separated, the gel sticks to IPC. Before use, the plates are cleaned with water and alcohol. Full-length clamps are slid over the glass plate sandwich assembly and secured by moving the levers towards the IPC. The bottom edge of the IPC assembly is placed into the precision caster base, secured by cam pegs and laid horizontally with the IPC facing up. The acrylamide mix is prepared and poured into the syringe which is connected to the base of the gel caster via tubing. The mix is then injected until it reaches the comb. Care must be taken so that the gel assembly is horizontal otherwise there will be leakage. When the gel has to be run, the plate assembly is removed from the caster base and placed on the tray and secured by stabilizer bar. The buffer (1X TBE) is poured and the gel is pre-run for 30 min at 1700 volts. We use PowerPac HV and the run is over in 2-3hours.
We have not encountered any major difficultly with the instrument. However, recently the black material used for covering the cathode appears to be of poor quality, as it ripped apart exposing the wires within.
The Sequi-GenŽ GT apparatus is recommended.
| Comment on this Review |
Comments? Questions? Discuss this review of Sequi-Gen-GT-From-Bio-Rad with the author of this review, Nidhi Vishnoi, and the rest of the Biocompare community.
|
Nidhi Vishnoi
Senior Research Fellow
Chemical Carcinogenesis
Advanced Centre for Treatment, Research and Education in Cancer (ACTREC), TMC
India
more articles...
