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EZ DNA Methylation-Direct Kit
D5021 / D5020
EZ DNA Methylation-Direct Kit
- Complete bisulfite conversion of DNA directly from blood, soft tissue, cells, FFPE samples, and LCM samples.
- Compatible with small sample inputs as few as 10 cells or 50 pg of DNA.
- DNA recovered by this bisulfite conversion kit is ideal for downstream analyses including PCR, endonuclease digestion, sequencing, microarrays, etc.
The EZ DNA Methylation-Direct kit is a bisulfite conversion kit that features reliable and complete DNA bisulfite conversion directly from blood, tissue, and cells without the prerequisite for DNA purification. The increased sensitivity of this bisulfite conversion kit makes it possible to amplify bisulfite-converted DNA from as few as 10 cells or 50 pg DNA. Recovered bisulfite-converted DNA is ideal for downstream analyses including PCR amplification, 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: 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: 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.
Q9: 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.
|D5002-4||M-Wash Buffer||24 ml||$30.00|
|D5001-4||M-Wash Buffer||6 ml||$12.00|
|D5002-5||M-Desulphonation Buffer||40 ml||$50.40|
|D5001-5||M-Desulphonation Buffer||10 ml||$18.00|
|D5005-2||M-Dilution Buffer-Gold||1.5 ml||$12.00|
|D5006-2||M-Dilution Buffer-Gold||7 ml||$12.00|
|D5005-3||M-Binding Buffer||30 ml||$24.00|
|D5001-6||M-Elution Buffer||1 ml||$12.00|
|D5002-6||M-Elution Buffer||4 ml||$12.00|
|D5006-3||M-Binding Buffer||125 ml||$69.60|
|D5020-7||M-Solubilization Buffer||4.5 ml||$22.80|
|D5021-7||M-Solubilization Buffer||18 ml||$33.60|
|D5020-8||M-Reaction Buffer||1 ml||$18.00|
|D5020-9||M-Digestion Buffer (2X)||4 ml||$12.00|
|D5021-9||M-Digestion Buffer (2X)||15 ml||$18.00|
|C1004-50||Zymo-Spin IC Columns||50 Pack||$58.30|
|C1001-50||Collection Tubes||50 Pack||$16.50|
|D5001-1||CT Conversion Reagent||1 tube for 10 Conversions||$12.00|