Properties & Usage
Unit Definition
One unit is defined as the amount of enzyme required to protect 100 ng FAM-labeled dsDNA in 30 minutes at 37°C in a total reaction volume of 20 µl against cleavage by MboI restriction endonuclease. Reaction Conditions
1X rCutSmart™ Buffer
Supplement with 640 µM S-adenosylmethionine (SAM)
Incubate at 37°C
1X rCutSmart™ Buffer
50 mM Potassium Acetate
20 mM Tris-acetate
10 mM Magnesium Acetate
100 µg/ml Recombinant Albumin
(pH 7.9 @ 25°C)
Control Protection Assay
One unit of EcoGII Methyltransferase is incubated with 100 ng of an 80 bp FAM abeled dsDNA substrate in 20 µl of 1X rCutSmart Buffer supplemented with 640 µM SAM, for 30 minutes at 37°C followed by 10 minutes inactivation at 65°C. 0.12 units of Thermolabile Proteinase K is subsequently added into reaction followed by 30 minutes incubation at 37°C and 10 minutes inactivation at 55°C. The extent of protection is determined by addition of 5 units of MboI restriction endonuclease followed by incubation at 37°C for 30 minutes and 20 minutes inactivation at 65°C. Results are analyzed by capillary electrophoresis analysis, and the relative abundances of cleaved and uncleaved DNA species ar used to quantitate protection. Storage Buffer
10 mM Tris-HCl
250 mM NaCl
1 mM DTT
0.1 mM EDTA
0.15% Triton® X-100
180 µg/ml BSA
50% Glycerol
pH 7.4 @ 25°C
Heat Inactivation
65°C for 10 minutes Unit Assay Conditions
EcoGII Methyltransferase is incubated with 100 ng of a FAM-labeled dsDNA substrate (80 bp) in 20 µl of 1X rCutSmart Buffer supplemented with 160 µM SAM, for 30 minutes at 37°C followed by 10 minutes at 65°C. The extent of protection is determined by addition of 5 units of MboI restriction endonuclease (incubation at 37°C for 30 minutes, 65°C for 20 minutes) followed by capillary electrophoresis analysis to resolve the reaction products, assign the cleaved and uncleaved species and quantitate their abundance.