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EZ DNA Methylation-Startup™ Kit

A complete system for DNA methylation detection: DNA bisulfite treatment, robust hot-start PCR, and a universally methylated human control DNA standard with primers.
Designed for the first time user requiring a consolidated product to perform DNA methylation analysis.

Product Size Catalog # Price Qty
EZ DNA Methylation-Startup™ Kit 50 Rxns D5024

About EZ DNA Methylation-Startup™ Kit

The EZ DNA Methylation-Startup™ Kit provides the necessary technologies required for complete bisulfite conversion of DNA for PCR and methylation analysis. This kit includes bisulfite conversion reagents that allow for direct sampling of blood, cells, and fresh or FFPE tissues without the prerequisite for upstream DNA purification (see EZ DNA Methylation-Direct™ Kit). A fully methylated Universal Methylated Human DNA Standard is provided together with a special primer set for PCR to control for and assess conversion efficiency. Finally, a unique ZymoTaq™ DNA Polymerase is included for robust amplification of bisulfite-treated DNA.

Learn More About Bisulfite Conversion Click to see which kit is right for you

Format Spin Column
DNA Recovery > 80%
Processing Time 4 hrs
Equipment Microcentrifuge
Binding Capacity 5 µg/prep
Elution Volume ≥ 10 µl
Conversion Efficiency > 99.5%
Input DNA, cells, blood, tissue, FFPE

For reagent preparation and DNA bisulfite treatment details, please see the EZ DNA Methylation-Direct™ Kit instructions. The following protocol uses the supplied Universal Methylated Human DNA Standard as an example and should be used as a guideline for your experimental samples.

Section I.  Bisulfite Conversion of DNA (please reference the EZ DNA Methylation-Direct™ Kit instruction manual as needed)

                        2 µl   Universal Methylated Human DNA Standard (or user supplied sample - see note below)
                        18 µl   H2O
                        20 µl   total volume

Note:  The maximum input volume of DNA is 20 µl.  Alternatively, cells and tissues can be input directly without the prerequisite for DNA purification (reference the EZ DNA Methylation-Direct™ Kit protocol).

CT Conversion Reagent to the DNA in the PCR tube.  Mix the sample and then centrifuge briefly to ensure no droplets are retained in the cap or the sides of the tube.EZ DNA Methylation-Direct™ Kit protocol through to the DNA elution step (i.e., Step 9).The eluted DNA will be in Elution Buffer (10 mM Tris-HCl, pH 8.5, 0.1 mM EDTA)  and can be used for PCR, microarrays, bisulfite sequencing, or other applications.

Section II.  PCR Amplification of Bisulfite-Treated DNA

The following PCR conditions have been optimized for amplification of the Universal Methylated Human DNA Standard following bisulfite treatment and should be used as a guideline when setting up your own PCR.  The control primers (i.e., hMLH1 I and II) are designed to specifically amplify a 182 bp product.  See Appendix I (page 6) for detailed information regarding the design and use of the Universal Methylated Human DNA Standard and Control Primers, see Appendix II for general information on bisulfite PCR.

                   12.5 µl   2X ZymoTaq™ PreMix
                        1 µl   hMLH1 Primer I        
                        1 µl   hMLH1 Primer II
                        2 µl   Bisulfite-Treated Universal Methylated Human DNA Standard
                     8.5 µl   ddH2O
                      25 µl   Total Volume

Note: The amount of input DNA in the PCR can be increased or decreased as needed. The final concentration of MgCl2 in the reaction is 1.75 mM. If required, adjust reaction volumes accordingly to optimize the MgCl2, primer, and/or template concentrations

Universal Methylated Human DNA Standard.Temperature      Time

        95°C           10 minutes
        95°C           30 seconds
        59°C           30 seconds         35-40 cycles
        72°C           60 seconds
        72°C           7 minutes
          4°C           “hold”

For amplification of user provided bisulfite-treated DNA, the annealing temperature and extension time should be adjusted according to the primer Tms and amplicon size, respectively.  We recommend using between 35-40 cycles for most templates.  Please refer to Appendix II for additional PCR and primer design guidelines.  The amplified PCR product can be used for gel analysis, TA cloning, restriction endonuclease digestion, microarrays, sequencing, and other downstream molecular applications.

For specific notes and additional information, please see the product protocol PDF.
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