Ammonium Acetate Solution

Applications

• Recovery of nucleic acids from LMP agarose gels for use in: restriction mapping; cloning; labeling; DNA sequencing;¹ transformation of YAC,^2,3 BAC,⁴ cosmid, fosmid, and plasmid vectors; microinjection;² and amplification.

GELase™ Agarose Gel-Digesting Preparation contains a unique β-agarose digesting enzyme developed at EPICENTRE for simple, quantitative recovery of intact DNA and RNA from low-melting point (LMP) agarose gels following electrophoresis in TAE, TBE, MOPS, or phosphate buffers. The gel may be digested directly in the electrophoresis buffers or GELase Buffer may be added to, or exchanged with, those buffers for higher activity. GELase Preparation digests the carbohydrate backbone of molten agarose, releasing small, soluble oligosaccharides. The nucleic acid can be used in the digested gel solution or precipitated using ammonium acetate/ethanol. The gel digestion products are alcohol-soluble.

Benefits

• Recoveries of DNA or RNA consistently approach 100% (Fig. 1).
• Purify even megabase DNA (Fig. 1) or RNA that is intact and biologically active.^1,5,6
• Simple, flexible protocol with minimal hands-on time.
• A typical 200-mg gel slice in TAE buffer can be digested in less than 10 minutes using only 3 units of GELase Preparation (Table 1).
• More active than other gel-digesting enzymes.
• More economical than spin columns or other gel-digesting methods.
• Available in two convenient concentrations: 1 U/µl for standard reactions and 0.2 U/µl for greater economy in digesting multiple small gel samples or extending digestion times.

Unit Definition: One unit of GELase Preparation digests 600 mg (~600 µl) of molten 1% LMP agarose gel in GELase Buffer in 1 hour at 45°C.

Storage Buffer: 50% glycerol containing 50 mM Tris-HCl (pH 7.5), 0.1 mM EDTA, 0.1 M NaCl, 0.1% Triton X-100, and 1 mM DTT.

Quality Control: Each lot of GELase Preparation is free of detectable DNA exonuclease, endonuclease, and RNase activities.

Notes:

1. One unit of GELase Preparation equals 3 or more units of most other gel-digesting enzymes.
2. We recommend adding ammonium acetate for nucleic acid precipitation because GELase Preparation and most other proteins are not precipitated by ethanol in the presence of ammonium acetate. Also, the solubilities of the oligosaccharide digestion products are higher in ethanol in the presence of ammonium acetate. For your convenience, a ready-to-use 5 M Ammonium Acetate Solution is available.

References

1. Kirkpatrick, H.A. et al. (1997) EPICENTRE Forum 4(3), 11.
2. Sasaki, H. and Hogan, B.L. (1994) Cell 76, 103.
3. Schedl, A. et al. (1993) Nature 362, 258.
4. Woo, S.S. et al. (1994) Nucleic Acids Res. 22, 4922.
5. Steck, T.R. (1994) BioTechniques 17, 676.
6. Chen, L. et al. (1994) BioTechniques 16, 228.

Figure 1. Recovery of any size DNA approaches 100% using GELase™ Gel-Digesting Preparation. In Experiment 1, DNAs from 63 bp to 7.9 kb were separated in a 1.5% LMP-agarose gel (A1), purified using GELase Preparation, and analyzed on a new, 1.5% agarose gel (B1) stained with ethidium bromide. In Experiment 2, high-molecular-weight soybean DNA was separated on a 1% LMP-agarose gel by pulsed-field electrophoresis (A2). The 2.2 Mb DNA band was purified using GELase Preparation, and analyzed on a new 1% LMP-agarose gel stained with ethidium bromide (B2). (Experiment 2 results courtesy of L. Chen and A. Atherly, Dept. of Zoology & Genetics, Iowa State Univ., Ames, IA.)

Table 1. Time required to digest 200 mg of 1% LMP agarose in TAE buffer.

Figure 2 (click to enlarge). GELase protocols are simple and flexible, providing highest activity or maximum speed.