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DNA Modifying Enzymes and Cloning >
Methyltransferases >
GpC Methyltransferase (M.CviPI) |
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Prices are in US dollars and valid only for US orders.
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Methylation Site:
Description:
The GC Methyltransferase, M.CviPI, methylates all cytosine residues (C5) within the double-stranded dinucleotide recognition sequence 5´...GC...3´.
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).
Applications:- Blocking restriction endonuclease cleavage
- Altering the physical properties of DNA
- Uniform [3H]-labeling of DNA
Reagents Supplied:
GC Reaction Buffer (10X)
S-adenosylmethionine (SAM) (200 X)
Enzyme Properties
Heat Inactivation:
65°C for 20 minutes
Reaction & Storage Conditions
Reaction Conditions:
1X GC Reaction Buffer
Supplemented with 160 μM S-adenosylmethionine (SAM)
Incubate at
37°C.
1X GC Reaction Buffer:
50 mM Tris-HCl
50 mM NaCl
10 mM Dithiothreitol
pH 8.5 @ 25°C
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.
Protection Assay Conditions:
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.
Concentration:
4,000 units/ml
Storage Conditions:
15 mM Tris-HCl
0.2 mM NaCl
1 mM Dithiothreitol
0.1 mM EDTA
200 µg/ml BSA
50% Glycerol
pH 7.4 @ 25°C
Storage Temperature:
-20°C
Notes
General 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, (1) 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).
Quality Control for Current Lot
Quality control values for a specific lot can be found on the datacard which accompanies each product.
Quality Assurance Statement:
Purified free of contaminating endonucleases and exonucleases.
Endonuclease Activity:
Incubation of a 50 μl reaction containing 40 units of GpC Methyltransferase (M.CviPI) with 1 μg of
ΦX174 RF I DNA for 4 hours at 37ºC resulted
in < 5% conversion to RFII as determined by agarose gel electrophoresis.
16-Hour Incubation::
Incubation of a 50 μl 1X GC Reaction Buffer containing 60 units of GPC Methyltransferase (M.CviPI) with 1 μg of λ DNA for 16 hours at 37°C resulted in no detectable endonuclease contamination.
Exonuclease Activity::
Incubation of a 50 μl GC Reaction Buffer containing 80 units of GpC Methyltransferase (M.CviPI) with 1 μg of a mixture of single and double-stranded [3H] E. coli DNA (105 cpm/μg) for 4 hours at 37°C released < 0.1% of the total radioactivity.
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.
Reagents Sold Separately
S-adenosylmethionine (SAM)
Legal
Patents:
New England Biolabs, Inc.: U.S. Patent Nos. 7,034,116, 6,492,168
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