EpiTect Methyl II DNA Restriction Kit

Description

Brief Protocol

Materials Included

The EpiTect Methyl II DNA Restriction Kit (12) 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 methylated 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 EpiTect Methyl II DNA Restriction Kit (12) together with the Epitect Methyl II PCR Arrays and Custom PCR 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 EpiTect II Methyl DNA Restriction Kit with the EpiTect Methyl II 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  660 µl × 4 Tubes 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 EpiTect Methyl II DNA Restriction Kit (12) 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: EpiTect Methyl II PCR Arrays EpiTect Methyl II PCR Primer Assays for individual gene promoters RT² SYBR® Green qPCR Master Mixes (Available for all real-time PCR instruments) The EpiTect Methyl II DNA Restriction Kit uses the MethylScreen™ Technology provided under license from Orion Genomics, LLC. Purchaser agrees that use of this product and data therefrom is limited solely to the purchaser and for only the purchaser’s own internal molecular biology research applications (“Permitted Use”), and shall not be re-sold or used for any other purposes (all of which are expressly prohibited), including without limitation diagnostic purposes, uses that could require regulatory approval for diagnostics from an agency of any government or regulatory entity anywhere in the world, diagnosis, prevention, or treatment of disease, and the right to perform commercial services of any kind, including without limitation, reporting the results of purchaser’s activities, including without limitation, for a fee or other commercial consideration. Except for the Permitted Use, no rights, titles, or interests in or to any tangible or intangible property rights are conveyed or shall be deemed conveyed by implication, estoppel or otherwise. The performance characteristics of the product other than for the Permitted Use are unknown. Back to Top