New England Biolabs, M0392L, β-Agarase I

Related Categories Other,, DNA Gel Extraction Applications Nucleic Acid Purification Specification Unit Definition One unit is defined as the amount of enzyme required to digest 200 μl of molten low melting point or NuSieve agarose to nonprecipitable neoagaro-oligosaccharides in 1 hour at 42°C. Reaction Conditions 1X β-Agarase I Reaction Buffer Incubate at 42°C 1X β-Agarase I Reaction Buffer 10 mM Bis-Tris-HCl 1 mM EDTA (pH 6.5 @ 25°C) Storage Buffer 50 mM Bis-Tris-HCl 1 mM EDTA 50% Glycerol pH 6.5 @ 25°C Heat Inactivation 65°C for 15 minutes FAQ Q: Can ligation and other DNA manipulations be carried out on β-Agarase I treated gel slices containing DNA? A: Yes. Q: What is the molecular weight of β-Agarase I? A: 30,000 Daltons Q: What type of agarose will β-Agarase I digest? A: Only low melting point agarose is suitable for β-Agarase I digestion as the solution must be liquid at the incubation temperature of 42°C. If the temperature falls below 42°C during the reaction time, even low melting point agarose will begin to congeal and be undigestable. Q: Will β-Agarase I work in other buffers? A: Yes. β-Agarase I will work in water and other buffers. The β-Agarase I concentration should be doubled for full activity in other buffers. The EDTA in the β-Agarase I buffer protects the DNA from a low level of nicking seen in buffers containing magnesium. Q: Why does a white precipitate form after the reaction using β-Agarase I? A: The reaction did not go to completion. See the questions below to optimize the reaction. Q: Can a high percentage low melt gel be digested with β-Agarase I? A: β-Agarase I works most efficiently on solutions containing 1% agarose or lower. For maximum digestion of higher percentage gels, melt the gel slice at 65°C and adjust the volume with 1X β-Agarase I buffer so that the final concentration of agarose is 1%. Q: What is a common cause of β-Agarase I reaction failure? A: Working at the wrong temperature. β-Agarase I is quickly inactivated at temperatures above 45°C. The low melting point agarose must be melted at 65°C then equilibrated at 42°C. Therefore, when working with large volumes, be sure to leave ample time for molten agar to equilibrate to 42°C. At lower temperatures the agarose starts to solidify making it resistant to digestion. Q: Does the gel running buffer have an effect on the β-Agarase I reaction? A: β-Agarase I works best on gels made with Tris-acetate buffer(TAE). For gels made with Tris-borate buffer (TBE), doubling the required amount of β-Agarase I is recommended. Q: How can β-Agarase I be heat inactivated? A: Incubation at 95°C for 2 minutes or incubation at 65°C for 15 minutes inactivates 50 units of β-Agarase I. Q: How stable is β-Agarase I in reaction? A: β-Agarase I retains activity for several hours at 45-50°C and is stabilized by the presence of agarose in the reaction. Q: What is the optimal pH for β-Agarase I digestion? A: β-Agarase I exhibits optimal activity at pH 6.5. Greater than 75% of the optimal activity is maintained between pH 5.0 - 8.5.