EZ DNA Methylation-Gold Kit
D5006 / D5005
EZ DNA Methylation-Gold Kit
- Complete bisulfite conversion of DNA in less than 3 hours.
- Desulphonation and recovery of bisulfite-treated DNA with a spin column.
- Recovered DNA is ideal for downstream analyses such as PCR, endonuclease digestion, sequencing, microarrays, etc.
The EZ DNA Methylation-Gold Kit is a refinement of our popular EZ DNA Methylation kit and uses heat denaturation instead of chemical denaturation of the input DNA. This allows for the denaturation and bisulfite conversion steps to be consolidated into one step, leading to a much-reduced incubation time. Recovered bisulfite-converted DNA is ideal for PCR amplification for downstream analyses including 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||Microcentrifuge and thermocycler with heated lid|
|Input||500 pg - 2 µg of DNA.|
|Processing Time||3 hours|
|Sample Source||Purified genomic DNA, endonuclease-digested DNA, linearized plasmid DNA, etc. DNA should be high-quality and RNA-free.|
Q1: Tips for bisulfite primer design?
Q2: 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.
Q3: 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.
Q4: 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).
Q5: What is the minimum DNA size that can be recovered?
> 50 bp.
Q6: How to quantify converted DNA?
For best results, keep the method of quantification consistent before and after bisulfite treatment:
- If quantifying with a NanoDrop, use dsDNA settings (50 μg/ml for Ab260 = 1.0) before treatment and use RNA settings (40 μg/ml for Ab260 = 1.0) after treatment.
- If quantifying with Qubit, use a dsDNA assay before treatment and use a ssDNA assay after treatment.
Q7: How to visualize converted DNA?
Following bisulfite treatment, DNA will be single stranded with limited non-specific base pairing at room temperature. To visualize, run the converted DNA on an agarose gel then chill the gel on ice or in an ice bath for 30 minutes. This will force enough base-pairing to allow intercalation of the ethidium bromide for the DNA to be visible. If using a Bioanalyzer or TapeStation instrument, use RNA kits and reagents to visualize the converted DNA.
Q8: 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.
Q9: How long is bisulfite converted DNA stable at -20 °C?
Converted DNA eluted in M-Elution Buffer can generally be stored at -20°C for 1-3 months. If longer term storage is necessary, we recommend storing at or below -70°C if possible. Bisulfite converted DNA is less stable than dsDNA; for best results, minimize freeze-thawing of converted DNA and use as soon as possible for downstream analysis.
“It makes gene specific DNA methylation analysis very simple and anyone with modest technical background can easily perform the experiment using this kit.”
- Murali B. (Centre for DNA Fingerprinting and Diagnostics)
“This protocol allows for bisulfite conversion in about 3 hours. This fast conversion is particularly relevant to our experiments, which allows us to PCR amplify and clone the products in 1 day, saving time without loss of quality.”
- Joseph M. (Sanford-Burnham Medical Research Institute)
|D5002-4||M-Wash Buffer||24 ml||€30,60|
|D5001-4||M-Wash Buffer||6 ml||€12,30|
|D5001-5||M-Desulphonation Buffer||10 ml||€18,40|
|D5002-5||M-Desulphonation Buffer||40 ml||€51,30|
|D5005-2||M-Dilution Buffer-Gold||1.5 ml||€12,30|
|D5006-2||M-Dilution Buffer-Gold||7 ml||€12,30|
|D5005-3||M-Binding Buffer||30 ml||€24,50|
|D5002-6||M-Elution Buffer||4 ml||€12,30|
|D5001-6||M-Elution Buffer||1 ml||€12,30|
|D5006-3||M-Binding Buffer||125 ml||€70,90|
|D5005-6||M-Dissolving Buffer||500 µl||€12,30|
|D5006-6||M-Dissolving Buffer||1.2 ml||€18,40|
|C1001-50||Collection Tubes||50 Pack||€16,80|
|C1004-50||Zymo-Spin IC Columns||50 Pack||€59,40|
|D5001-1||CT Conversion Reagent||1 tube for 10 Conversions||€12,30|