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5-Azadc Treated Jurkat Genomic DNA |
 | | | | 5-Azadc Treated Jurkat Genomic DNA | | | |
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Prices are in US dollars and valid only for US orders.
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Description:
Genomic DNA purified from human male Jurkat (human acute T-cell leukemia) cells that are treated with 5-aza-2-deoxycytidine (5-Azadc), suitable as a negative control in the study of CpG dinucleotide methylation in the genome.
Source:
Jurkat (acute T-cell leukemia) cells were grown to 50 % confluency in RPMI plus 10% fetal bovine serum and were treated with a 2 µM 5-aza-2'-deoxycytidine for eight days. Genomic DNA was isolated by a standard genomic purification protocol (1), phenol/chloroform extracted and equilibrated to 10 mM Tris-HCl (pH 7.5) and 1 mM EDTA.
Applications:- A negative 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:- A260/280 Ratio: 1.90
- The potent methyltransferase inhibitor (MTI) 5-aza-2-deoxycytidine (5-Azadc) (Decitabine, Dacogen) causes growth arrest, differentiation, and/or apoptosis of many cell types in vitro and in vivo. The genomic DNA derived from cells treated with this drug exhibit some lower molecular weight smearing when visualized on a 0.8% agarose gel. Significant (up to 70%) genome-wide CpG demethylation was confirmed by bisulfite sequencing of IGS ribosomal DNA (rDNA).
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 protein and RNA.
Bisulfite Sequencing:
10 µl (1 µg) of 5-Azadc treated Jurkat Genomic DNA and normal Jurkat Genomic DNA were bisulfite converted (3) and eluted in 40 µl of TE buffer. 5 µl were added to a 20 µl PCR reactions containing primers specific to the fully CpG methylated intergenic spacer (IGS) ribosomal DNA (rDNA). 30% of the CpG dinucleotides normally methylated in the control DNA this region were demethylated in the 5-Azadc treated Jurkat Genomic DNA as determined from DNA sequenced from the appropriate sized PCR products.
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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