Unit Definition

One unit is defined as the amount of enzyme required to protect 1 µg (dam-) Lambda DNA in 1 hour at 37°C in a total reaction volume of 10 µl against cleavage by MboI restriction endonuclease.

Reaction Conditions

1X dam Methyltransferase Reaction Buffer
Supplement with 80 µM S-adenosylmethionine (SAM)
Incubate at 37°C

1X dam Methyltransferase Reaction Buffer
50 mM Tris-HCl
5 mM β-ME
10 mM EDTA
(pH 7.5 @ 25°C)

Control Protection Assay

dam Methyltransferase is incubated with 1 µg of (dam-) Lambda DNA in 10 µl of 1X dam Methyltransferase Buffer, supplemented with 80 µM S-adenosylmethionine, for 1 hour at 37°C followed by 15 minutes at 65°C. The extent of protection is determined by addition of 40 µl 1X NEBuffer 3 supplemented with 10 mM MgCl₂ and 10 units of MboI restriction endonuclease. Incubation at 37°C for 1 hour is followed by analysis on an agarose gel.

Storage Buffer

50 mM Tris-HCl
50 mM KCl
1 mM DTT
10 mM EDTA
200 µg/ml BSA
50% Glycerol
pH 7.5 @ 25°C

Heat Inactivation

65°C for 20 minutes