Unit Definition

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

Reaction Conditions

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

1X HaeIII Methyltransferase Reaction Buffer
50 mM Tris-HCl
50 mM NaCl
10 mM DTT
(pH 8.5 @ 25°C)

Control Protection Assay

HaeIII Methyltransferase is incubated with 1 µg of λ DNA in 10 µl of 1X HaeIII Methyltransferase Reaction Buffer, supplemented with 80 µM S-adenosylmethionine, for one hour at 37°C followed by 15 minutes at 65°C. The extent of protection is determined by the addition of 40 µl NEBuffer 2.1 and 10 units of HaeIII restriction endonuclease. Incubation for 1 hour at 37°C is followed by analysis on an agarose gel.

Storage Buffer

50 mM Tris-HCl
1 mM DTT
10 mM EDTA
200 µg/ml BSA
50% Glycerol
50 mM KCl
pH 7.4 @ 25°C

Heat Inactivation

65°C for 20 minutes