EZ DNA Methylation-Gold Kit

Protocol tips

Upstream tips	Protocol tips	Downstream tips
	Always include fully methylated (in-vitro methylated DNA) and non-methylated controls (Leucocyte DNA, DNMT1/3 double KO DNA) during MSP

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

Genomic DNA from cells and bone tissue samples was isolated with phenol:chloroform: isoamyl alcohol (Invitrogen, 15593031). For optimized bisulfite conversion, we used 1000 ng of genomic DNA and the Zymo EZ DNA Methylation-Gold kit (Zymo Research, D5005). After bisulfite conversion, the DNA was bound to a Zymo spin column and desulfonated. The bisulfite converted DNA was eluted from the column in 10μl of elution buffer. In the qMSP reaction, duplicate aliquots of 1μl of the eluted bisulfite-treated genomic DNA were amplified with AmpliTaq Gold DNA Polymerase buffer (Applied Biosystems, N808–0240) with 2 mM MgCl2, 0.4 pmol primers, 400 μM dNTP mix, 8 μl of Sybr Green and 1U of AmpliTaq Gold DNA polymerase in a total volume of 25 μl. Amplification conditions were: 95°C for 5 min, followed by 40 cycles of 95°C for 30s, 57°C for 30s and 72°C for 30s, with a final extension of 72°C for 5 min. Epitec methylated and unmethylated human control DNA, bisulfite converted, as well as unmodified human DNA (Qiagen, 59695) were used as controls to check primer specificity (Fig. 1). The degree of DNA methylation was estimated as the unmethylated primer cycle threshold minus the methylated primer cycle threshold for each amplicon. Thus, smaller values are associated with smaller proportions of methylated DNA in the sample. The mean and SD was calculated from series of three experiments for each amplicon and cell line.

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Papers

Check out relevant papers found by Labettor's AI that are relevant for performing DNA methylation profiling Gene specific profiling - MG-63 RANKL using EZ DNA Methylation-Gold Kit from Zymo Research.

Manufacturer protocol

Download the product protocol from Zymo Research for EZ DNA Methylation-Gold Kit below.

Download manufacturer protocol

Videos

Check out videos that might be relevant for performing DNA methylation profiling Gene specific profiling - MG-63 RANKL using EZ DNA Methylation-Gold 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.