DNA Methylation Enzyme Kit

Description

Brief Protocol

Materials Included

The Methyl-Profiler DNA Methylation Enzyme Kit prepares your genomic DNA samples for analysis by real-time PCR primers and arrays for known and predicted methylated CpG islands by real-time PCR. Using the enzymes and buffer provided in the kit, four digests are performed to detect different methylated DNA fractions. The product of a mock digest contains all of the input genomic DNA. The product of the methylation-sensitive restriction enzyme (Enzyme A) digest contains hypermethylated DNA sequences, while the product of the methylation-dependent restriction enzyme (Enzyme B) digest contains unmethylated DNA sequences. The product of a double digest measures the background and the success of both enzymatic digestions. Use the DNA Methylation Enzyme Kit together with the Methyl-Profiler™ DNA Methylation PCR Arrays and Custom qPCR Primers to analyze the methylation status of CpG islands in the promoter of your genes or other methylated DNA sequences of interest.

Description

Brief Protocol

Materials Included

For Experienced Users Only. First time users of the Methyl-Profiler DNA Methylation Enzyme Kit with the DNA Methylation PCR Arrays should first refer to the User Manual. Thaw the 5X Digestion Buffer and dissolve any cotton-like precipitate by vortexing briefly. Dilute each genomic DNA sample into a DNase-free 1.5-mL tube using the appropriate recipe listed in the User Manual for the gene list and format of your chosen PCR Array. Mix well by vortexing, and briefly centrifuge to the bottom of the tube. Set up four digestion reactions in separate PCR tubes using the appropriate recipe listed in the User Manual for the gene list and format of your chosen PCR Array. NOTE: All four tubes must contain the same amount of genomic DNA. Pipette up and down to thoroughly, yet gently, mix the components. Spin tubes briefly in microcentrifuge. Incubate at 37 ºC for 6 hours in a thermal cycler. Inactivate the enzymes at 65 ºC for 20 minutes following the digestion. Store DNA digests at -20 ºC until used. Spin down before use. Back to Top

Description

Brief Protocol

Materials Included

Please check the kit components immediately after you receive this package. We are not responsible for any missing items not reported within two (2) business days upon receipt. Tube / Contents  Quantity 5X Restriction Digestion Buffer  300 µl in One 1.5-mL Tube Enzyme A (Methylation-Sensitive)  25 µl in One 0.5-mL Tube Enzyme B (Methylation-Dependent)  25 µl in One 0.5-mL Tube Storage Conditions: The DNA Methylation Enzyme Kit is shipped either on dry ice or with blue ice packs and must be stored at -20 ºC. We suggest that all kit contents be kept in their original container to insure that no components are misplaced. Shelf Life: All reagents are stable for 6 months after receipt of the kit if stored at the recommended temperature. Related Products: Methyl-Profiler DNA Methylation PCR Arrays Methyl-Profiler DNA Methylation qPCR Primers for individual gene promoters RT² SYBR® Green qPCR Master Mixes (Available for all real-time PCR instruments) SYBR® is a registered trademark of Invitrogen. RT² and Methyl-Profiler™ are trademarks of SABiosciences. Back to Top