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|
|DNA Recovery||> 80%|
|Processing Time||4 hrs|
|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)
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).
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.
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
95°C 10 minutes
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.