Gla I

6,000.00 24,000.00 

  • CNY: 466.92 ¥ - 1,867.68 ¥

MD DNA endonuclease Gla I.
The enzyme cleaves C5-methylated DNA and doesn’t cut unmodified DNA.

  • Recognition site: R(5mC)↑GY / YG↓(5mC)R
  • Source: An E.coli strain that carries the cloned Gla I gene from Glacial ice bacterium Gl29
  • Optimal buffer: SE-buffer Y
  • Optimal temperature: 30
  • Inactivation temperature, 20 minutes at: 65
SKU: SE-E493 Category: Tag:

Available Options

SKUРackagePackageConcentrationPrice 
E493S1000 u5 000 u/ml6,000.00 
  • CNY: 466.92 ¥
E494L5000 u5 000 u/ml24,000.00 
  • CNY: 1,867.68 ¥

Description

Recognition site and hydrolysis position:

R(5mC)GY R(5mC)↑GY
YG(5mC)R YG↓(5mC)R

Source: An E.coli strain that carries the cloned Gla I gene from Glacial ice bacterium Gl29
Substrate specificity:
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
Lanes:
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.
GlaI activity assay on DNA pHspAI2/GsaIIn the presence of 20% DMSO, the enzyme’s activity significantly increases without losing specificity

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 5`-G(5mC)G(5mC)-3`/3`-(5mC)G(5mC)G-5` [2].
Optimal SE-buffer: SE-buffer Y
Enzyme activity (%):

B G O W Y ROSE
75 75 25 25 100 100

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.
Ligation: –
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.Reagents Supplied with Enzyme

10 X SE-buffer Y, pHspAI2/GsaI DNA
Methylation sensitivity: The enzyme cleaves only C5-methylated DNA and does not cut unmodified DNAand DNA with N4-methylcytosines [1].
Notes:

References:

  1. Chernukhin V.A., Nayakshina T.N., Tomilova J.E., Mezentseva N.V., Dedkov V.S., Degtyarev S.Kh. Bacterial strain Glacial ice bacterium I – producer of GlaI restriction endonuclease. // Russian Federation patent RU 2287012 C1 (2006).
  2. Valery A. Chernukhin, Tatyana N. Najakshina, Murat A. Abdurashitov, Julia E. Tomilova, Nina V. Mezentzeva, Vladimir S. Dedkov, Natalya A. Mikhnenkova, Danila A. Gonchar, Sergei Kh. Degtyarev A novel restriction endonuclease GlaI recognizes methylated sequence 5’-G(m5C)^GC-3’ // Biotechnologia (russ.). 2006. N 4. P. 31-35
  3. Degtyarev S.Kh., Belavin P.A., Repin V.E., Malygin E.G. Preparation method of restriction endonuclease Fok I from Flavobacterium okeanokoites // Soviet Union patent SU1436160 (1988). (In Russian)
  4. Degtyarev S.Kh., Rechkunova N.I., Grinev A.A., Dedkov V.S. Discovery and substrate specificity determination of restriction endonucleases Bme18I and Kzo9I.// Izvestia SB SA USSR, Biological sciences series, No.3, 25-26 (1989). (In Russian)
  5. Zemlyanskaya, E.V., Degtyarev, S.K. Substrate specificity and properties of methyl-directed site-specific DNA endonucleases.// Molecular Biology, v.47, No.6, p.900-913 (2013)

Application:

 

Additional Information