EZ DNA Methylation-Direct 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 (MSP)
DNA bisulfite modification and purification were performed using an EZ DNA methylation-Direct kit (Zymo Research Corporation, Irvine, California, USA) according to the instruction. Concentration of DNA was evaluated by absorbance at 260 nm on a UV spectrophotometer (BioRad Inc., Hercules, CA, USA). The sets of primers for miR-30a/c-5p gene were flanking the 3kb regions of the 5’ upstream regions from the start of pre-miR-30a/c-5p sequences. The primers for methylation-specific PCR were designed by MethPrimer and the sequences were as follows: miR-30a methylated (M)-forward (F): 5’-TAAATATTTTAAGGATAGAGAGCGA-3’, miR-30a methylated (M)-reverse (R): 5’-AAAATTTAAAAACAACCCGAA-3’, miR-30a unmethylated (U)- forward (F): 5’-AAATATTTTAAGGATAGAGAGTGA-3’, miR-30a unmethylated (U)- reverse (R): 5’-ATAAAAAATTTAAAAACAACCCAAA-3’; miR-30c methylated (M)-forward (F): 5’-AGGCGTTTGTAGTTTTAGTTATTCG-3’, miR-30c methylated (M)-reverse (R): 5’-CGAAATAAAATCTCGCTCTATCG-3’, miR-30c unmethylated (U)- forward (F): 5’-GTGTTTGTAGTTTTAGTTATTTGG-3’, miR-30c unmethylated (U)- reverse (R): 5’-TATACTCCAACTACTCACACCACG-3. DNA amplification was performed with Epi Taq HS (Takara Biotechnology Co. Ltd., Dalian, China) under the following condition: 94 °C for 5 min; 40 cycles of 94 °C for 45 s, 44.3 °C (miR-30a U)/48 °C (miR-30a M)/36 °C (miR-30c U)/50 °C (miR-30c M)/for 45 s, 72 °C for 1 min; and 72 °C for 10 min. The PCR products were separated by 2.0 % agarose gel electrophoresis and visualized by a chemiluminescence imaging system (Bio-Rad, Richmond, CA, USA).

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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 - A2780 miR-30a-5p using EZ DNA Methylation-Direct Kit from Zymo Research.

Manufacturer protocol

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

Download manufacturer protocol

Videos

Check out videos that might be relevant for performing DNA methylation profiling Gene specific profiling - A2780 miR-30a-5p using EZ DNA Methylation-Direct 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.