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DNA Modifying Enzymes and Cloning >
Methyltransferases >
Human DNA (cytosine-5) Methyltransferase (Dnmt1) |
 | | | | Human DNA (cytosine-5) Methyltransferase (Dnmt1) | | | |
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Prices are in US dollars and valid only for US orders.
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Methylation Site:
Description:
Dnmt1 methylates cytosine residues in hemimethylated DNA at 5'...CG...3' (1,2). Mammalian Dnmt1 is believed to be involved in carcinogenesis, embryonic development and several other biological functions (3-5). The bulk of the methylation takes place during DNA replication in the S-phase of the cell cycle (6).
Source:
Dnmt1 is expressed from human Dnmt1 cDNA using a baculovirus expression system (1,7).
Reagents Supplied:
Dnmt1 Reaction Buffer (10X)
BSA
S-adenosylmethionine (SAM)
Enzyme Properties
Heat Inactivation:
65°C for 20 minutes
Reaction & Storage Conditions
Reaction Conditions:
1X Dnmt1 Reaction Buffer
Supplemented with 100 μg/ml Bovine Serum Albumin and 160 μM S-adenosylmethionine (SAM)
Incubate at
37°C.
1X Dnmt1 Reaction Buffer:
50 mM Tris-HCl
1 mM Dithiothreitol
1 mM EDTA
5 % Glycerol
pH 7.8 @ 25°C
Unit Definition:
One unit is the amount of enzyme required to catalyze the transfer of 1 pmol of methyl group to poly dI.dC substrate in a total reaction volume of 25 μl in 30 minutes at 37°C.
Concentration:
2,000 units/ml
Storage Conditions:
50 mM Tris-HCl
200 mM NaCl
1 mM Dithiothreitol
1 mM EDTA
50% Glycerol
pH 7.5 @ 25°C
Storage Temperature:
-20°C
Notes
General notes:- Storage of SAM: S-adenosylmethionine is stored at –20°C as 32 mM solution dissolved in sulfuric acid (0.005 M) and 10% ethanol. SAM in this solution stored under ideal conditions remains active for up to 6 months. SAM is unstable at (pH 7.5), 37°C, and should be replenished for reactions incubated longer than 4 hours.
Many problems in achieving complete digestion can be alleviated by using fresh SAM.
- For DNA modification and protection applications, M.SssI (NEB #M0226) is preferred because it efficiently methylates both unmethylated and hemimethylated DNA substrates.
FAQs
- Is S-adenosylmethionine (SAM) supplied with the Methyltransferase?
- What source of tritiated SAM is recommended for use with Dnmt1?
- What should be considered if the methylation using Dnmt1 is not going to completion?
- How does Dnmt1 differ from SssI methylase?
- What is the specific activity of Dnmt1?
- Can Dnmt1 be heat inactivated?
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.
References
- Pradhan, S. et al. (1999) J. Biol. Chem., 274, 33002-33010.
- Bacolla, A. et al. (1999) J. Biol. Chem., 274, 33011-33019.
- Schmutte, C. et al. (1998) Biol. Chem., 379, 377-388.
- Laird, P.W. et al. (1995) Cell, 81, 197-205.
- Li, E. et al. (1992) Cell, 12, 915-926.
- Leonhardt, H. et al. (1992) Cell, 71, 865-873.
- Yen, R.W. et al. (1992) Nucl. Acids Res., 20, 2287-2291.
Reagents Sold Separately
BSA
S-adenosylmethionine (SAM)
Companion Products
Human DNA (cytosine-5) Methyltransferase (Dnmt1) Amino-terminal Ab
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