GpC Methyltransferase (M.CviPI)

Description

The GC Methyltransferase, M.CviPI, methylates all cytosine residues (C5) within the double-stranded dinucleotide recognition sequence 5´...GC...3´.

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:

Store at (°C)Concentration
GC Reaction Buffer10X
S-adenosylmethionine (SAM)-2032 mM

Advantages and Features

Applications

  • Blocking restriction endonuclease cleavage
  • Altering the physical properties of DNA
  • Uniform [3H]-labeling of DNA

Properties and 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

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 Temperature

-20°C

Storage Conditions

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 min

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.
  1. Requires fresh DTT for optimum activity. For best results, mix assay buffer fresh for each use.
  2. 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).

References

  1. Zacharias, W. et al. (1988). Biochemistry. 27, 2970-2978.
  2. Gruenbaum, Y. et al. (1982). Nature. 295, 620-621.
  3. Murchie, A.I. and Lilley, D.M. (1989). J. Mol. Biol.. 205, 593-602.
  4. Ohmori, H. et al. (1978). Nucl. Acids. Res. 5, 1479-1485.
  5. Xu, S. et al. (1998). Nucl. Acids Res.. 26, 3961-3966.
  6. Kladde, M.P. et al. (1991). Methods Enzymol. . 304, 431-447.

FAQs

  1. Will GpC Methyltransferase (M. CviPI) work in CutSmart Buffer?
  2. Will any NEB methyltransferases (methylases) work on RNA?

Tech Tips

Make sure the reaction buffer is diluted out just before doing the methylation reaction. This enzyme requires fresh DTT for optimal activity.

Datacards

The Product Summary Sheet, or Data Card, includes details for how to use the product, as well as details of its formulation and quality controls. The following file naming structure is used to name the majority of these document files: [Catalog Number]Datasheet-Lot[Lot Number]. For those product lots not listed below, please contact NEB at info@neb.com or fill out the Technical Support Form for appropriate document.

Interactive Tools

Quality Control

Quality Assurance Statement

  • Purified free of contaminating exonucleases and endonucleases.

Quality Control Assays

The following Quality Control Tests are performed on each new lot and meet the specifications designated for the product. Individual lot data can be found on the Product Summary Sheet/Datacard or Manual which can be found in the Supporting Documents section of this page. Further information regarding NEB product quality can be found here.
  • Endonuclease Activity (Nicking):
    The product is tested in a reaction containing a supercoiled DNA substrate. After incubation for 4 hours the percent converted to the nicked form is determined by agarose gel electrophoresis.
  • Exonuclease Activity (Radioactivity Release):
    The product is tested in a reaction containing a radiolabeled mixture of single and double-stranded DNA. After incubation for 4 hours the exonuclease activity is determined by the % release of radioactive nucleotides.
  • Non-Specific DNase Activity (16 hour, Buffer):
    The buffer is tested in a reaction containing a DNA substrate. After incubation for 16 hours there is no detectable degradation of the DNA substrate as determined by agarose gel electrophoresis.

Safety Data Sheet

The following is a list of Safety Data Sheet (SDS) that apply to this product to help you use it safely.

Datacards

The Product Summary Sheet, or Data Card, includes details for how to use the product, as well as details of its formulation and quality controls. The following file naming structure is used to name the majority of these document files: [Catalog Number]Datasheet-Lot[Lot Number]. For those product lots not listed below, please contact NEB at info@neb.com or fill out the Technical Support Form for appropriate document.