Gla I

Supplied in:

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.

Reaction Conditions:

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.

Unit Definition:

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’ .

Substrate specifity: 

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 /

5`-A(5mC)GT-3`/3`-TG(5mC)A-5`.

Medium substrates ( > 6% activity): 5`-G(5mC)R(5mC)-3`/3`-(5mC)GYG-5` /

5`-G(5mC)GT-3`/3`-CG(5mC)A-5`.

Bad substrates (6% activity): 5`-G(5mC)GC-3`/3`-CG(5mC)G-5`.

Quality Assay:

16-Hour Incubation:

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.

Oligonucleotide Assay:

No detectable degradation of a single- and double-stranded oligonucleotide was observed after incubation with 8 units of enzyme for 3 hours.

Enzyme Properties 

Activity in SEBuffers:

SEBuffer B    75-100%

SEBuffer G    75-100%

SEBuffer O    75-100%

SEBuffer W    75-100%

SEBuffer Y     75-100%

SEBuffer ROSE   100%

Reagents supplied with enzyme:

10XSEBuffer Gla I, DNA pHspAI2/GsaI.

Heat Inactivation:

Yes (65°C for 20 minutes)