3% TREVIGEL PROTOCOL for 3 bp resolution

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Burns 1998
USDA Forest Service Research, Rhinelander, WI, USA

The agarose-type gel we use for screening SSR PCR products is 3% Trevigel 500 (Trevigen, Cat# 9804-250-P). If done correctly, 3 bp resolution can be obtained by running the gel in 1X TAE at 5 volts/cm for 2 hours with a 40 cm gel.

For SSR marker screening in 20 x 40 cm gels:

For 400 ml of gel solution: At room temperature, stir 350 ml 1X TAE buffer in a 1 Liter screw cap bottle while sprinkling in 12 g Trevigel (to keep from clumping). Rehydrate at room temperature for 10-15 min. Add 1X TAE to bring total volume to 400 ml. A 1 Liter bottle should be used so the gel will not boil out of the bottle.

: The 1X TAE used to make the gel must come from the same 1X TAE stock used as a running buffer for the gel. Differences in ionic concentration will occur if different stocks or dilutions are used, and you will get poor results.

To dissolve the Trevigel, heat in a microwave at 100% power for 5 min. Using insulated gloves, add 4ul of 10mg/ml ethidium bromide and swirl the solution several times to help the gel dissolve and to mix the EtBr. Heat gel solution at 100% power for 3 min with lid of bottle loose. Add dH2O to bring the volume above 400 ml and heat the gel in the microwave again until enough water has boiled off to bring the volume back to 400 ml.

Place the bottle in sink and quickly degas with a water aspirator. When bubbles start appearing at the surface, wait a few moments and then remove the black stopper from the bottle before turning off the water.

Quickly pour the gel in a 40 cm tray without the combs. (Don"t wait for the solution to cool to 55 C.) The gel does not flow easily so you must start at one end of the tray and quickly pour the gel to the other end of the tray. Carefully insert the combs and let gel set up at least 15 min.

: Having EtBr present in the gel during electrophoresis will cause unpredictable alterations in relative mobilities of some fragments. As the EtBr does not appear to affect resolution, we live with the quirky migrations to save time. The alternative is to equilibrate the gel for a couple of hours with EtBr post-electrophoresis.

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