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EZ-96 DNA Methylation-Direct Kit
D5023 / D5022
EZ-96 DNA Methylation-Direct Kit
- High-throughput 96-well 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 DNA.
- DNA recovered by this bisulfite conversion kit is ideal for downstream analyses including PCR, endonuclease digestion, sequencing, microarrays, etc.
The EZ-96 DNA Methylation-Direct Kit is a 96-well 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 96-well 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 PCR amplification for downstream analyses including restriction 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||Shallow well: > 30 µl
Deep-well: >15 µl
|Equipment||Thermocycler with heated lid, swinging-bucket centrifuge with plate carriers.|
|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 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.
|D5021-9||M-Digestion Buffer (2X)||15 ml|
|D5021-7||M-Solubilization Buffer||18 ml|
|D5006-3||M-Binding Buffer||125 ml|
|D5007-6||M-Elution Buffer||8 ml|
|D5006-2||M-Dilution Buffer-Gold||7 ml|
|D5003-1||CT Conversion Reagent (96 Conversions)||1 Bottle|
|D5002-5||M-Desulphonation Buffer||40 ml|
|D5007-4||M-Wash Buffer||36 ml|
|C2003||Elution Plate||2 Plates|
|C2002||Collection Plate||2 Plates|
|C2005||Conversion Plate w/ Cover Foil||2 Plates/Foils|
|C2004||Zymo-Spin I-96 Plate||2 Plates|
|C2001||Silicon-A Plate||2 Plates|