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.	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

Bisulfite Sequencing
Bisulfite modification of genomic DNA was performed using either the Epitech (Qiagen, Valencia, CA) or EZ DNA Methylation (Zymo, Irvine, CA) kits. MethPrimer was used to design primers to specifically amplify photoreceptor specific genes and the putative promoter regions of two intergenic miRNAs ( and ) (, , ). For direct sequencing, the PCR products were agarose gel-purified and directly sequence by the High-Throughput Genomics Unit at the University of Washington using the forward primers. Using the ContigExpress function of Vector NTI Advance 10 (Invitrogen, Carlsbad, CA), the chromatograph peak heights for C and T at each CpG site were measured and the % methylation (C peak height/(T peak height+C peak height)) versus CpG position, with respect to the TSS, was plotted . For sequencing of individual clones after PCR amplification, the presence of a specific PCR product was confirmed by agarose gel electrophoresis, and the PCR product for each set of primers was subcloned using the TOPO-TA cloning kit (Invitrogen, Valencia, CA). Thirty sequences (10 clones for each of 3 biological replicates) with ≥95% conversion of the non-CpG cytosines were obtained and sequenced using the M13 reverse primer by the University of Washington High-throughput Genomics Unit. Fasta files of each sequence were analyzed using a custom JAVA program (DNAMethylMap, unpublished software by B. Kumar and S. Yegnasubramanian).

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Reviews

Discussion

Papers

Check out relevant papers found by Labettor's AI that are relevant for performing DNA methylation profiling Gene specific profiling - HEK 293T RBP3 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 - HEK 293T RBP3 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.