Imprint® DNA Modification Kit

Protocol tips

Upstream tips	Protocol tips	Downstream tips
For best results, the CT Conversion Reagent should be prepared freshly and used immediately following preparation.	Always include fully methylated (in-vitro methylated DNA) and non-methylated controls (Leucocyte DNA, DNMT1/3 double KO DNA) during MSP

Upstream tips
For best results, the CT Conversion Reagent should be prepared freshly and used immediately following preparation.
Protocol tips
Always include fully methylated (in-vitro methylated DNA) and non-methylated controls (Leucocyte DNA, DNMT1/3 double KO DNA) during MSP

Publication protocol

Genomic DNA Isolation and Sodium Bisulfite Modification

The total genomic DNA from White Blood Cells (WBCs) and from cell lines has extracted using the Qiagen Genomic DNA Isolation Kit (Qiagen Inc. FlexiGene DNA kit, Germany) and the DNA concentration and quality have determined using a spectrophotometer. After the determination of concentration of each DNA sample with spectrophotometry, sodium bisulfite modification has performed using the Imprint DNA Modification Kit (Sigma-Aldrich, Inc. UK) according to the manufacturer's protocol with minor adjustments. DNA has used immediately or should be stored at -20°C. Results from different sequences of methylated and unmethylated DNA bisulfite treatment has followed by MSP as described by Herman et al. [20].

Methylation Specific PCR (MSP) Assay

Analysis of the methylation status of bax gene promoter region has done by Methylation-Specific Polymerase Chain Reaction (MSP) assay, as described before [21]. This method is highly specific for the analysis of methylation of CpG dinucleotides in a CpG island. In brief, DNA methylation patterns of the CpG island existing in promoter sequence of the bax gene have determined by chemical modification of unmethylated but not the methylated cytosines to uracil and subsequent PCR using primers specific for either methylated or the modified unmethylated DNA. MSP has performed in a thermal cycler (Verriti, ABI, USA) with the following cycling conditions: The amplifications have consisted of a Taq activation step at 95°C for 5min followed by 35 amplification cycles (95°C for 30 s, annealing temperature for 45 s, and 72°C for 30 s) and final incubation at 72°C for 5min. The PCR mixture contained 100-200 ng of bisulfite-treated DNA, 1×PCR Buffer (CINAGEN), 1.5 mM MgCl2, 0.2 mM of each dNTP, 20 pmol of each primer set, and 1.25 units of Taq (Sinagen Inc., Tehran, Iran) in a final volume of 25 μl. Amplified products have electrophoresed on 2.5% agarose gels, stained by ethidium bromide and directly visualized under Ultraviolet (UV) illumination. All MSP assays have repeated at least twice. The primer sets for MS-PCR of bax gene have listed in Table 1 [21].

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Manufacturer protocol

Download the product protocol from Sigma-Aldrich for Imprint® DNA Modification Kit below.

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