10 mM Tris-HCl (pH 7.5); 200 mM NaCl; 0,1 mM EDTA; 0.05% Triton X-100; 100 µg/ml BSA; 7 mM 2-mercaptoethanol; 50% glycerol.
1XSEBuffer Gla I
Incubate at 30°C.
1XSEBuffer Gla I (pH 8.5 @ 25ºC)
10 mM Tris-HCl, 10 mM NaCl, 5 mM MgCl2, 1mM 2-mercaptoethanol.
One unit is defined as the amount of enzyme required to hydrolyse completely a unique 5`-G(5mC)G(5mC)-3`/3`-(5mC)G(5mC)G-5` site in 1 µg of pHspAI2
plasmid DNA, which is linearized with GsaI, in 1 hour at 30ºC in a total reaction volume of 50 µl. Concentrated enzymes are diluted to approximately 1000 units/ml with the buffer [10 mM Tris-HCl
(pH 7.6); 50 mM KCl; 0.1 mM EDTA; 1 mM DTT; 200 g/ml BSA; 50% glycerol] before the activity determination. DNA pHspAI2/GsaI is a linearized plasmid pHspAI2, which carries a gene of
DNA-methyltransferase M.HspAI (recognition sequence 5'-GCGC-3') and includes a unique GlaI recognition site 5’-G(5mC)G(5mC)-3’/3’-(5mC)G(5mC)G-5’ .
The enzyme activity depends on number and position of methylated nucleotides in the recognition sequence:
Optimal substrate (100% activity ): 5`G(5mC)G(mC)-3`/3` (m5C)G(m5C)G-5`.
Good substrates ( > 25% activity): 5`-R(5mC)G(5mC)-3`/3`-YG(5mC)G-5 /
Medium substrates ( > 6% activity): 5`-G(5mC)R(5mC)-3`/3`-(5mC)GYG-5` /
Bad substrates (6% activity): 5`-G(5mC)GC-3`/3`-CG(5mC)G-5`.
No detectable degradation of 1 μg of Lambda DNA was observed after incubation with 8 units of enzyme for 16 hours at 30°C in a total
reaction volume of 50 μl.
No detectable degradation of a single- and double-stranded oligonucleotide was observed
after incubation with 8 units of enzyme for 3 hours.