EZ DNA Methylation 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.

Protocol tips
For best results, the CT Conversion Reagent should be prepared freshly and used immediately following preparation.

Publication protocol

DNA methylation analysis
Bisulphite PCR-Luminex (BPL) methylation analysis was performed as described [18]. PCR primers sets, biotinylated at their 5'-end, were designed for gene amplification. PCR reaction mix contained 0.2 μM primer, 0.2 mM dNTPs, 1 × PCR buffer (50 mM KCl, 10 mM Tris-HCl, pH 8.3). 3 mM MgCl2, 2% dimethyl sulfoxide (DMSO), 0.625U Taq DNA Polymerase (Roche, Tokyo, Japan) and 100-200 ng of bisulphite treated DNA in a total volume of 25 μl. PCR conditions: 40 cycles of 95°C for 20 s/60°C for 30 s/72°C for 30 s using a GeneAmp 9700 thermal cycler (Applied Biosystems, CA, USA). Oligonucleotide probe sequences (Additional file 2: Table S1 in Ref 18) were synthesized and covalently bound to carboxylated fluorescent microbeads using ethylene dichloride (EDC). These oligonucleotide-labeled microbeads (oligobeads) were mixed together to make an oligobeads mixture of 100 oligobeads/μl and hybridized to the 5'-biotin-labeled PCR amplicons in a total volume of 50 μl per well in a 96-well plate by adding 5 μl of the appropriate oligobead mixture and 5 μl of the PCR amplicons to 40 μl of hybridization buffer. This reaction mixture was first denatured at 95°C for 2 min and then hybridized at 48°C for 30 min. After hybridization, the oligobeads were washed in 100 μl of PBS-Tween and pelleted by microcentrifugation. Pelleted oligobeads were reacted with a 70 μl aliquot of a 100 × diluted solution of SA-PE in PBS-Tween. Hybridized amplicons were labeled with SA-PE at 48°C for 15 min. Reaction outcomes were measured by the Luminex 100 flow cytometer. Methylation assays were additionally performed for each DMR using the conventional bisulphite treatment PCR methylation assay and combined bisulphite PCR restriction analysis (COBRA) as described previously [21].



Full paper   Login or join for free to view the full paper.

Reviews

Discussion

Papers

Check out relevant papers found by Labettor's AI that are relevant for performing DNA methylation profiling Gene specific profiling - SKOV3 PEG1 using EZ DNA Methylation kit from Zymo Research.

Manufacturer protocol

Download the product protocol from Zymo Research for EZ DNA Methylation kit below.

Download manufacturer protocol

Videos

Check out videos that might be relevant for performing DNA methylation profiling Gene specific profiling - SKOV3 PEG1 using EZ DNA Methylation kit from Zymo Research. Please note that these videos are representative and steps or experiment specific processes must be kept in mind to expect desired results.