5M Ammonium Acetate Solution

Catalogue number

Pack size

G1005ML

5ml

G1025ML

5 x 5ml

GELase™ 50X Reaction Buffer

Catalogue number

Pack size

G191ML

1ml

G195ML

5ml

GELase™ Agarose Gel-Digesting Prep - 0.2 U/µl

Catalogue number

Pack size

G31050

50 units

G31200

200 units

GELase™ Agarose Gel-Digesting Prep - 1U/µl

Catalogue number

Pack size

G09050

50 units

G09100

100 units

G09200

200 units

GELase™ Agarose Gel-Digesting Preparation is a unique enzyme solution 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.
GELase Preparation digests the carbohydrate backbone of molten agarose into small, soluble oligosaccharides. The purified nucleic acid can be rapidly recovered from the digested gel solution by ammonium acetate/ethanol precipitation.

Unit Definition:

One unit of GELase™ Preparation digests 600mg of molten 1%LMP-agarose gel in GELase™ Buffer in 1 hour at 45°C.

Storage Buffer:

50% glycerol containing 50mM Tris-HCl, pH 7.5, 0.1mM EDTA, 0.1M NaCl, 0.1% Triton® X-100, and 1mM DTT.

Quality Control:

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

We recommend using 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 5M Ammonium Acetate Solution is available.

Time to digest 200mg of 1% LMP Agarose in TAE Buffer

units of GELase™ Prep	Digestion Time
3.0	8 min
2.0	15 min
1.0	30 min
0.33	60 min

Last modified: 2008-05-14 16:46:46

Applications

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

Benefits

• High recovery - yields consistently approach 100%
• Purify any size nucleic acid - from oligos to multi-megabase DNA^1,5 or RNA
• Gentle procedure⁶ - nucleic acid is purified intact and with high biological activity
• Fast - a typical 200mg gel slice can be digested in less than 10 minutes using only 3 units of GELase™ Preparation
• Simple, flexible protocol, minimal hands-on time
• Two convenient concentrations - 1U/µl for standard reactions and 0.2U/µl for greater economy in digesting multiple small gel samples or extending digestion times
• High activity - GELase preparation is more active than other gel-digesting enzymes

Unit Definition:

One unit of GELase Preparation digests 600mg (~600µl) of molten 1% LMP-agarose gel in GELase Buffer in 1 hour at 45°C. Note: One unit of GELase™ Preparation equals 3 or more units of most other gel-digesting enzymes.

Table 1. Time required to digest 200mg of 1% LMP agarose in TAE buffer.
Units of GELase	Digestion Time
3.0	8 min.
2.0	15 min.
1.0	30 min.
0.33	60 min.

Storage Buffer:

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

Quality Control:

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

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). Migration and staining intensity indicate near-quantitative recovery of each DNA, from <100bp to multi-megabase size, without degradation using GELase Preparation. (Experiment 2 results courtesy of L. Chen and A. Atherly, Dept. of Zoology & Genetics, Iowa State Univ., Ames, IA.)

Figure 2. GELase protocols are simple and flexible, providing highest activity or maximum speed.

*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.

Last modified: 2008-05-14 16:46:46

Protocols for: GELase™ Agarose Gel-Digesting Preparation

GELase™ Agarose Gel-Digesting preparation / GELase™ 50X Reaction Buffer Protocol

(catalogue number G09050 / G09100 / G09200 / G31050 / G31200 / G191ML / G195ML)

5M Ammonium Acetate Solution Protocol

(catalogue number G1005ML / G1025ML)

Please note: all protocols off site are the responsibility of the products supplier

Last modified: 2008-05-14 16:46:46

References

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

Last modified: 2008-05-14 16:46:46