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

2.8. Bisulfite Modification of Genomic DNA
DNA was modified by method described by Clark et al. [23]. Briefly, 2 μg of genomic DNA was denatured by adding 2 μL of 3 M NaOH. Samples were incubated at 37°C for 15 minutes and then at 90°C for 2.5 minutes. The tubes were immediately placed in ice so that DNA remains denatured. The tubes were centrifuged at 8000 rpm for 10 seconds to settle DNA. For each reaction, modification mixture was prepared by mixing: denatured DNA (2 μg), 208 μL, saturated sodium metabisulphite, pH-5, and 10 mM hydroquinone. Samples were properly mixed and incubated at 55°C in water bath for 16 hours.

The modified DNA was then desulfonated and purified using Sigma’s Imprint DNA Modification Kit (MOD50) using the prescribed protocol by the manufacturer. The modified DNA was then stored at −20°C until use.

2.9. Methylation-Specific PCR (MS-PCR)
MS-PCR was performed using primers specific for methylated and unmethylated DNA for GSTP1, p16, p14, and RASSF1A genes. Primers were designed using Methprimer, a program for designing bisulfite-conversion-based Methylation PCR Primers (http://www.urogene.org/methprimer/index1.html) [24] from the promoter regions of the four genes whose sequences were retrieved from Transcriptional Regulatory Element Database, TRED (http://rulai.cshl.edu/cgi-bin/TRED/tred.cgi?process=home) [25].

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Manufacturer protocol

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

Download manufacturer protocol

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