EpiTect MSP 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

Methylation specific PCR for promoter of MEG3
First, DNA from cells and tissues was extracted using the QIAmp DNA Mini kit (Qiagen, German), according to the manufacturer’s recommendations. Next, we used the EpiTect Bisulfite kit (Qiagen) for bisulfate conversion following the product manual. At last, Methylation specific PCR (MSP) was performed on Bisulfite-treated DNA using EpiTect MSP kit (Qiagen). The primers of MEG3 promoter which specific for methylated and unmethylated were as follows: the methylated pair (M) was 5′-GTT AGT AAT CGG GTT TGT CGG C (forward) and 5′-AAT CAT AAC TCC GAA CAC CCG CG (reverse); the unmethylated pair (U) was 5′-GAG GAT GGT TAG TTA TTG GGG T (forward) and 5′-CCA CCA TAA CCA ACA CCC TAT AAT CAC A (reverse). The PCR reaction was conducted in DNA Engine Tetrad 2 Peltier Thermal Cycler (Bio-Rad) under the following conditions: 95 °C 15 min; 94 °C 30s, 70 °C 30s, 72 °C 30s, 5 cycles; 94 °C 30s, 65 °C 30s, 72 °C 30s, 5 cycles; 94 °C 30s, 60 °C 30s, 72 °C 30s, 30 cycles; 72 °C 7 min. The PCR products were identified by electrophoresis through 2.5% agarose gel, stained with ethidium bromide, visualized under UV and compared by densitometry.

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Papers

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Manufacturer protocol

Download the product protocol from Qiagen for EpiTect MSP Kit below.

Download manufacturer protocol

Videos

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