b-Agarase Code J683 ; b-Agarase Code J682

Directions for Use

AMRESCO’s b-Agarase is free of contaminating RNase activity.

Unit Definition: 1 U of b-Agarase will solubilize 200 mg of molten 0.5% Agarose II per hour at 40-42°C.

Note: The unit definition is based on a 0.5% gel in 1X TAE. Increases in agarose concentration or electrophoresising in other buffers will require more enzyme to obtain complete hydrolysis. (Example: A 2% Agarose II gel in 1X TAE requires 4 U per 200 mg; a 1% Agarose SFR gel in 1X TBE requires 7-10 U per 200 mg.)

• After isolating the band of interest, equilibrate the agarose block against 1X Reaction Buffer (J714, supplied), for a final volume of 10X in an appropriately sized tube. (A 1.5 ml microcentrifuge tube works well.) Next, 1 hour of gentle rocking at 22°C should be performed. The buffer can then be removed.

• Incubation at 68°C in a waterbath for 15-20 minutes is normally required to completely melt the agarose. This is checked visually by tapping the reaction tube with a finger. (Gels run in TBE generally require more time to melt.)

• Transfer the tube to a 42°C waterbath or heating block. Incubate for 5 minutes. Add 10X Reaction Buffer to a final concentration of 1X. Mix by tapping and continue to incubate for 2 minutes at 42°C in the waterbath or with the heating block.

• Aliquot the appropriate amount of enzyme into the tube. Tap the tube with a finger to mix. Incubate for 1 hour at 42°C in a waterbath or with a heating block. (Note: Activity can be increased by raising the temperature to 45°C for 1 hour.)

• To avoid precipitation of un-digested agarose, the investigator may choose to centrifuge the reaction tube at 12,000 rpm for 30 seconds and transfer the solubilized agarose to a new microcentrifuge tube.

• Add 7M ammonium acetate to a final concentration of 2.5M. Tap with finger to mix. Pipette 2-3X the reaction volume of ethanol into the tube. Mix.

• Allow to precipitate for 30 minutes to 1 hour at 22°C (room temperature). Centrifuge for 1 hour at 12,000-14,000 rpm at 22°C. Wash pellet with 70% ethanol and repeat spin.

• Remove as much of the ethanol as possible without disturbing the pellet. This can be done by pippetting off the supernatant and tipping the tube to a 45° angle on a benchtop covered with clean filter paper or a paper towel (to reduce contamination). Allow the pellet to dry.

• Re-suspend the pellet in the required volume of TE, pH 7.5, or appropriate buffer.

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