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GpC Methyltransferase (M.CviPI)

Catalog # Concentration Size List Price Quantity Your Price
M0227L 4000 units/ml 1000 units $506.00
$455.40
M0227S 4000 units/ml 200 units $125.00
$112.50
Catalog # Size List Price Your Price
M0227L 1000 units $506.00
$455.40
M0227S 200 units $125.00
$112.50
Catalog #
Qty:
 
*On-line ordering is for Canadian customers only. Web pricing is applicable only to orders placed online at www.neb.ca

  • Methylates all cytosine residues (C5) within the double-stranded dinucleotide recognition sequence 5´...GC...3´.
  • Blocks restriction enzyme cleavage
  • Altering the physical properties of DNA
  • Uniform (3H)-labeling of DNA
Product Source
The GpC Methyltransferase, M.CviPI, is isolated from a strain of E. coli which contains the methyltransferase gene from Chlorella virus. This construct is fused to the maltose binding protein (MBP).
Reagents Supplied

The following reagents are supplied with this product:

NEB # Component Name Component # Stored at (°C) Amount Concentration
  GpC Methyltransferase (M.CviPI) M0227SVIAL -20 1 x 0.05 ml 4,000 units/ml
  GC Reaction Buffer B0227SVIAL -20 1 x 1.5 ml 10 X
  S-adenosylmethionine (SAM) B9003SVIAL -20 1 x 0.1 ml 32 mM
  GpC Methyltransferase (M.CviPI) M0227LVIAL -20 1 x 0.25 ml 4,000 units/ml
  GC Reaction Buffer B0227SVIAL -20 1 x 1.5 ml 10 X
  S-adenosylmethionine (SAM) B9003SVIAL -20 1 x 0.1 ml 32 mM
Application Features
  • Blocking restriction endonuclease cleavage
  • Altering the physical properties of DNA
  • Uniform [3H]-labeling of DNA

Properties & Usage

Unit Definition

One unit is defined as the amount of enzyme required to protect 1 µg of λ DNA in a total reaction volume of 20 µl in 1 hour at 37°C against cleavage by HaeIII restriction endonuclease. 

Reaction Conditions

1X GC Reaction Buffer
Supplement with 160 µM S-adenosylmethionine (SAM)
Incubate at 37°C

1X GC Reaction Buffer
50 mM NaCl
50 mM Tris-HCl
10 mM DTT
(pH 8.5 @ 25°C)

Control Protection Assay

M.CviPI is incubated with 1 µg λ DNA in 20 µl 1X GC Reaction Buffer and 160 µM S-adenosylmethionine, for one hour at 37°C. The extent of protection by M.CviPI is determined by the addition of 30 µl NEBuffer 2 containing 10 units of HaeIII restriction endonuclease. Incubation for 1 hour at 37°C is followed by analysis on an agarose gel.

Storage Buffer

15 mM Tris-HCl
0.2 M NaCl
1 mM DTT
0.1 mM EDTA
200 µg/ml BSA
50% Glycerol
pH 7.4 @ 25°C

Heat Inactivation

65°C for 20 minutes

Materials Sold Separately
Notes
    • S-adenosylmethionine (SAM) is supplied as a 32 mM solution in 0.005 M sulfuric acid and 10% ethanol. Under these conditions SAM is stable for up to 6 months when stored at -20°C.
    • SAM is unstable at (pH 7.5), 37°C, and should be replenished in reactions incubated longer than 4 hours.
  • Requires fresh DTT for optimum activity. For best results, mix assay buffer fresh for each use.
  • Methylation at cytosine residues has also been shown to affect the physical properties of DNA, including lowering the free energy of Z-DNA formation (1), increasing the helical pitch of DNA (2), and altering the kinetics of cruciform extrusion (3). Positions of 5-methylcytosine can be identified due to decreased reactivity to hydrazine in chemical sequencing protocols (4).
  • MgCl2 is not required as a cofactor.
References
  • Zacharias, W. et al. (1988). Biochemistry. 27, 2970-2978.
  • Gruenbaum, Y. et al. (1982). Nature. 295, 620-621.
  • Murchie, A.I. and Lilley, D.M. (1989). J. Mol. Biol.. 205, 593-602.
  • Ohmori, H. et al. (1978). Nucl. Acids. Res. 5, 1479-1485.
  • Xu, S. et al. (1998). Nucl. Acids Res.. 26, 3961-3966.
  • Kladde, M.P. et al. (1991). Methods Enzymol. . 304, 431-447.
FAQs
Tech Tips
  • Make sure the reaction buffer is diluted out just before doing the methylation reaction. This enzyme requires fresh DTT for optimal activity.
Quality Control Assay
Quality Control tests are performed on each new lot of NEB product to meet the specifications designated for it. Specifications and individual lot data from the tests that are performed for this particular product can be found and downloaded on the Product Specification Sheet, Certificate of Analysis, data card or product manual. Further information regarding NEB product quality can be found here.
Specifications
The Specification sheet is a document that includes the storage temperature, shelf life and the specifications designated for the product. The following file naming structure is used to name these document files: [Product Number]_[Size]_[Version]
Certificate of Analysis
The Certificate of Analysis (COA) is a signed document that includes the storage temperature, expiration date and quality controls for an individual lot. The following file naming structure is used to name these document files: [Product Number]_[Size]_[Version]_[Lot Number]
Safety Data Sheets
Legal And Disclaimer

Products and content are covered by one or more patents, trademarks and/or copyrights owned or controlled by New England Biolabs, Inc (NEB). The use of trademark symbols does not necessarily indicate that the name is trademarked in the country where it is being read; it indicates where the content was originally developed. The use of this product may require the buyer to obtain additional third-party intellectual property rights for certain applications. For more information, please email busdev@neb.com.

This product is intended for research purposes only. This product is not intended to be used for therapeutic or diagnostic purposes in humans or animals.

New England Biolabs (NEB) is committed to practicing ethical science – we believe it is our job as researchers to ask the important questions that when answered will help preserve our quality of life and the world that we live in. However, this research should always be done in safe and ethical manner. Learn more.Licensed under U.S. Patent 7,034,116 and 6,492,168.
Licensed under U.S. Patent 6,492,168 and 7,034,116

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