EZ DNA Methylation-Startup Kit
EZ DNA Methylation-Startup Kit
- The complete solution for bisulfite conversion. This all-in-one kit contains reagents for bisulfite conversion of DNA without the need for purification, methylated human DNA with control primers, and a robust hot-start PCR polymerase that is specifically formulated for bisulfite-converted DNA.
- Designed for the first-time user requiring a consolidated product to control for bisulfite conversion.
- Kit includes: one EZ DNA Methylation-Direct kit, one fully methylated Universal Methylated Human DNA Standard, and ZymoTaq DNA Polymerase.
The EZ DNA Methylation-Startup Kit provides the necessary tools for complete bisulfite conversion of DNA for methylation analysis. This kit includes reagents that allow for bisulfite conversion of purified DNA and DNA directly from blood, cells, and fresh or FFPE tissues without the prerequisite for upstream DNA purification. A fully methylated Universal Methylated Human DNA Standard is provided with a special primer set for PCR to assess conversion efficiency. Finally, a unique ZymoTaq DNA Polymerase is included for robust amplification of bisulfite-treated DNA.
|Applicable For||Recovered DNA is ideal for downstream analyses including PCR, endonuclease digestion, sequencing, microarrays, etc.|
|Applications||Purified, converted DNA is of high-quality and well-suited for downstream processes, including library preparation for Next-generation sequencing, PCR amplification, etc.|
|Elution Volume||≥ 10 µl|
|Equipment||Thermocycler with heated lid and microcentrifuge.|
|Input||Samples containing between 50 pg to 2 µg of DNA. For optimal results, the amount of input DNA should be from 200 to 500 ng. The number of cells per standard treatment can range from 10-105 cells.|
|Processing Time||4 hours|
|Sample Source||Blood, tissue, cells, FFPE, LCM-derived samples, purified genomic DNA, endonuclease-digested DNA, linearized plasmid DNA, etc.|
Q1: What leads to poor conversion efficiency/ low yields?
Poor conversion efficiency and low yields can be due to a variety of different experiment-specific conditions. Please contact Technical Support to discuss your specific experimental conditions and further troubleshoot with a product specialist.
Q2: How to quantify / visualize converted DNA?
Following bisulfite treatment of genomic DNA, nonmethylated cytosine residues are converted into uracil. The recovered DNA is typically A, U, and T-rich. The original base-pairing no longer exists. Instead, it is single stranded with limited non-specific base-pairing at room temperature. The absorption coefficient at 260 nm resembles that of RNA. Use a value of 40 μg/ml for Ab260 = 1.0 when determining the concentration of the recovered bisulfite-treated DNA. To visualize, run converted DNA on agarose gel then chill the gel on ice for 30 minutes. The expected smear will be between 100-1500bp.
Q3: What is the minimum DNA size that can be recovered?
> 50 bp.
Q4: How long is bisulfite converted DNA stable at -20 °C?
Converted DNA should be eluted in M-Elution Buffer to keep the converted DNA stable for long term storage. If stored properly for long term (<-20C), the samples should last longer than a month. Minimize freeze/thawing to keep the bisulfite converted DNA stable.
Q5: Does bisulfite conversion only occur in a CpG context?
Bisulfite conversion will work regardless of context, so the kits are compatible with genomic DNA derived from plants and other species with high non-CpG methylation levels.
Q6: Is an incubation with desulphonation buffer for longer than 20 minutes recommended?
Leaving the desulphonation buffer on the column longer than recommended will cause more degradation and subsequently result in lower yields.
Q7: Which polymerase is recommended for amplification from bisulfite-converted DNA?
ZymoTaq DNA Polymerase has been specifically designed for use in bisulfite amplification reactions. ZymoTaq is available as a stand-alone polymerase (E2001/E2002), PreMix (E2003/E2004), and qPCR PreMix (E2054/E2055).
Q8: Tips for bisulfite primer design?
|E2003||ZymoTaq PreMix||(50 Rxns.) (2 x 625 µl)||$73.00|
|D5011||Universal Methylated Human DNA Standard||Human||$207.00|
|D5020||EZ DNA Methylation-Direct Kit||50 Rxns.||$193.00|