Gel cassettes

The transfer apparatus and the fiber pad used to hold the gel in the gel cassette should be thoroughly cleaned with detergents between use. Dirty fiber pads are a major cause of blot contamination. Also do not re-use transfer buffer, prepare fresh solution... 

The gel cassette is assembled according to manufacturer s instructions. Make sure that the glass plates are clean. If necessary, clean the plates with a common washing liquid, rinse thoroughly with water, and dry. Use plain spacers. Sealing of the cassette with self-adhesive tape, molten 1% agarose, or a small amount of polymerization mixture should be done only in exceptional cases. 

The gel cassette is mounted into the electrophoresis apparatus and electrophoresis tanks are filled with electrode buffer (Soln. H). Remove air bubbles at the interfaces between electrode buffer and... 

Solutions are gently mixed according to Table 2.9 in the indicated order and filled up with ddH20. Start the polymerization by addition of Soln. F and pour the mixture immediately into the gel cassette. When the appropriate height is reached, cover the liquid with water or n-butanol to get a smooth surface. Prepare the stacking gel as short as possible before starting the electrophoresis to avoid a decrease of the pH jump between stacking and separation gel by diffusion. 

Remove gel sandwich from casting stand and install it in the electrophoresis apparatus according to manufacturer s instructions. Make sure bottom of gel cassette is free of bubbles. 

Disassemble gel cassettes, place gels in separate covered trays, and immerse one gel in 50 ml of 2% casein in 50 mM Tris-Cl for 30 min at 4°C. Stain second gel with Coomassie brilliant blue as described (see Support Protocol 1, steps 1 to 5). 

Fill the transfer tank with lx transfer buffer. Submerge the gel cassette holder, fiber pads, and polyethylene sheet (after initial hydration as described in step 1) in transfer buffer. Place them inside the tank or submerge in transfer buffer using a separate tray. 

The final concentrations in the monomer solution delivered to the cassette are (5%T, 3.3%C), 5% glycerol, 2% ampholytes, 0.015% APS, 0.03% TEMED, 5 /zg/mL FMN. Position a fluorescent lamp about 3-4 cm from the gel cassette and illuminate the gel solution for about an hour. Gels may be used immediately or covered in plastic wrap and stored at 4°C for several days. Best results are sometimes obtained by letting a gel cure overnight at 4°C before use. 

Reusable Gel Cassettes and Membrane-Mediated Sample Loading... 

Gel electrophoresis apparatus (Owl Scientific or equivalent) and blank glass gel cassettes. Use 15 x 15-cm2 gels and 1.5-mm-thick combs and spacers for large-scale purification. Otherwise, use 0.75-mm combs and spacers or smaller (the thinner the gel, the higher the yield of gel-purified RNA). 

Fig. 15. Assembly of a gel cassette for casting IPGs, (a) Glass base plate (3 mm thick) (b) Gel Bond PAG foil (c) U-gasket (in general silicone or rubber, often glued to the glass cover plate usually 0.5 mm thick) (d) Template 3 mm thick glass plate with pieces of self-adhesive Dymo tape (250 p.m thick) for moulding of gel slots (e) paper clips as temporary, additional spacers. (From Gorg et al., 1978. Reproduced with permission of the publisher.)...

Place one or more gel cassettes onto the scan area. 

Remove gel cassettes from chamber, and using a plastic wedge, carefully open the cassette see Note 29). 

REUSABLE GEL CASSETTES AND MEMBRANE-MEDIATED SAMPLE LOADING... 

Cast vertical SDS-PAGE gels and prepare the electrophoresis chamber as described (see article by Julio E. Celis, Gitte Ratz, Bodil Basse, Jette B. Lauridsen, and Ariana Celis). Support the SDS gel cassette in a vertical position to facilitate the application of the first-dimension IPG gel strip. 

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