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CpG Methylated NIH 3T3 Mouse Genomic DNA |
 | | | | CpG Methylated NIH 3T3 Mouse Genomic DNA | | | |
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Prices are in US dollars and valid only for US orders.
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Description:
NIH 3T3 (mouse embryonic fibroblast cell line) genomic DNA that was enzymatically methylated with CpG Methylase (M.SssI), suitable as a positive control in the study of CpG dinucleotide methylation.
Source:
NIH 3T3 (mouse embryonic fibroblasts) cells were grown to confluency in DMEM plus 10% fetal bovine serum. Genomic DNA was isolated by a standard genomic purification protocol (1), treated with CpG Methylase (M.SssI), phenol extracted and equilibrated to 10 mM Tris-HCl (pH 7.5) and 1 mM EDTA.
Applications:- A positive control for Methylation-Specific PCR (MSP) (2), Bisulfite sequencing, Methylation-sensitive Single-Nucleotide Primer Extension (Ms-SNuPE), Combined Bisulfite Restriciton Analysis (COBRA), Bisulfite treatment and PCR-Single-Strand Conformation Polymorphism Analysis (Bisulfite-PCR-SSCP/BiPS).
Reaction & Storage Conditions
Concentration:
100 μg/ml
Storage Conditions:
10 mM Tris-HCl
1 mM EDTA
pH 7.5 @ 25°C
Storage Temperature:
-20°C
Avoid repeated freeze/thaw cycles.
Notes
General notes:- A 260/280 Ratio: 1.90
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 proteins and RNA.
Bisulfite conversion followed by Methylation-Specific PCR (MSP):
10 µl (1 µg) of CpG Methylated NIH 3T3 Mouse Genomic DNA were bisulfite converted (3) and eluted in 40 µl of TE buffer. 5 µl were added to a 20 µl PCR reaction containing primers specific to fully CpG methylated PTEN or Rb promoter DNA. A control set of primers designed to anneal to unmethylated PTEN or Rb promoter DNA were also used. Only the methylated-specific primer sets generated the appropriate sized PCR product.
S-adenosyl-L-[ methyl-3H] methionine (AdoMet) Incorporation Assay:
Incubation of 1 µg of CpG Methylated NIH 3T3 Mouse Genomic DNA with 4 µl 3H AdoMet, and 8 units of CpG Methylase (M.SssI) for 4 hours at 37°C in 50 µl of 50 mM Tris-HCl (pH 7.8), 1mM EDTA and 1 mM dithiothreitol incorporated 0.01% of the total radioactivity.
References
- Sambrook, J. and Russell, D. (2001) Molecular Cloning: A Laboratory Manual, (3rd ed.), (pp. 6.4-6.12). Cold Spring Harbor: Cold Spring Ha.
- Herman,J. G. and Baylin, S. B. (1996) U.S. Patent No, 5,786,146, Johns Hopkins University School of Medicine.
- Frommer, M., et. al. (1992) PNAS USA, 89, 1827-1831.
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