EpiTect Bisulfite 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

DNA was isolated with the DNeasy Blood and Tissue kit (Qiagen), and exactly 100 ng of genomic DNA prepared from the indicated cells were used for bisulfite modification with the EpiTect Bisulfite kit (Qiagen) according to the manufacturer’s instructions. Bisulfite-treated DNA was subjected to nested PCR with methylation-specific primers for the human H19-IGF2 DMR (32): 1st outer primer hH19O forward, AGG TGT TTT AGT TTT ATG GAT GAT GG and reverse, TCC TAT AAA TAT CCT ATT CCC AAA TAA CC; 2nd inner primer hH19I forward, TGT ATA GTA TAT GGG TAT TTT TGG AGG TTT and hH19-DMR-I reverse, same as hH19-DMR-O reverse. Next, the PCR products were cut with BstUI restriction enzyme and visualized on an agarose gel. In parallel after agarose gel electrophoresis, the PCR products were eluted using the QIAquick Gel Extraction kit (Qiagen). The eluted amplicons were subsequently ligated into the pCR2.1-TOPO vector and transformed into TOP10 bacteria using the TOPO TA Cloning kit (Invitrogen Life Technologies, Carlsbad, CA, USA). The plasmids were prepared using the QIAprep Spin Miniprep kit (Qiagen), sequenced with M13 forward and reverse primers, and the methylation pattern visualized using CpGviewer software. All experiments were conducted with three independent isolations of all cell populations.

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Discussion

Papers

Check out relevant papers found by Labettor's AI that are relevant for performing DNA methylation profiling Gene specific profiling - RH30 rhabdomyosarcoma IGF2 using EpiTect Bisulfite Kit from Qiagen.

Manufacturer protocol

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

Download manufacturer protocol

Videos

Check out videos that might be relevant for performing DNA methylation profiling Gene specific profiling - RH30 rhabdomyosarcoma IGF2 using EpiTect Bisulfite Kit from Qiagen. Please note that these videos are representative and steps or experiment specific processes must be kept in mind to expect desired results.