Source: An E.coli strain that carries the cloned Gla I gene from Glacial ice bacterium Gl29
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 30°C in a total reaction volume of 50 μl. As a result of this site hydrolysis two main DNA fragments (3,419 and 699 bp) are produced (see lanes 3-5 in the figure). GlaI digestion of recognition sequences with three and two 5-methylcytosines results in additional bands appearance (lane 6 in the figure).
|GlaI activity assay on DNA pHspAI2/GsaI
2 Control pHspAI2/GsaI DNA
3 – 0.5 μl GlaI (diluted 1/100),
4 – 1 μl GlaI (diluted 1/100),
5 – 2 μl GlaI (diluted 1/100),
6 – 1 μl of undiluted GlaI,
1 and 7- 1 Kb SE DNA Ladder.
Products were separated in 1% agarose gel in TAE Buffer.
Assayed on: 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
Optimal SE-buffer: SE-buffer Y
Enzyme activity (%):
Storage conditions: 10 mM Tris-HCl (pH 7.6); 250 mM NaCl; 0.1 mM EDTA; 7 mM 2-mercaptoethanol; 0,1% Triton X-100, 0.05 mg/ml BSA, 50% glycerol; Store at -20°C.
Non-specific hydrolisis: No detectable degradation of 1μg of Lambda DNA was observed after incubation with 100 units of enzyme for 16 hours at 30°C in a total reaction volume of 50 μl.
Methylation sensitivity: The enzyme cleaves only C5-methylated DNA and does not cut unmodified DNAand DNA with N4-methylcytosines .
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|E493||S||1000 u||5,500.00 ₽|
|E494||L||5000 u||22,000.00 ₽|