Highlights
- 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.
Description
The D5021 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
Applicable For
Purified, converted DNA is of high-quality and well-suited for downstream processes, including library preparation for Next-generation sequencing, PCR amplification, etc.
Conversion
> 99.5%
Elution Volume
? 10 ?l
Equipment
Thermocycler with heated lid and microcentrifuge.
Input
Samples containing between 50 pg to 2 ?g 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
Recovery
> 80%
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?
http://www.zymoresearch.com/bisulfite-beginner-guide https://www.zymoresearch.com/bisulfite-primer-seeker
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 / 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 ?g/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.
Q7: 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.
Q8: 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.
Single Cell
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99 / 100 Bioz Stars |
The integrated genomic landscape of thymic epithelial tumors
Cancer Cell – Published 12 Feb 2019
The Illumina Infinium HM450 array ( ) was used to assay 117 TCGA TET samples using standard protocols.. Briefly, genomic DNA (1000 ng) for each sample was treated with sodium bisulfite, recovered using the Zymo EZ DNA methylation kit (Zymo Research, Irvine, CA) according to the manufacturer?s specifications and eluted in 18 ul volume.. After passing quality control, bisulfite-converted DNA samples were whole genome amplified followed by enzymatic fragmentation and hybridized overnight
DNA methylation patterns separate senescence from transformation potenti…
Cancer Cell – Published 12 Feb 2019
Genomic DNA quality was assessed by low concentration agarose gel (0.6%) electrophoresis and spectrometry of OD260/280 and OD 260/230 ratio.. DNA bisulfite conversion was carried out using EZ DNA Methylation Kit (Zymo Research) by following manufacturer?s manual with modifications for Illumina Infinium Methylation Assay.. Briefly, 0.5 ? 1.0 ug of genomic DNA was first mixed with 5 ul of M-Dilution Buffer and incubate at 37?C for 15 minutes and then mixed with 100 ul
TETs Regulate Proepicardial Cell Migration through Extracellular Matrix …
Cell Rep – Published 07 Feb 2019
Peak calling and analysis of read density in peak regions were performed by macs14 1.4.2 with default parameters ( ).. Bisulfite sequencing was performed using the EZ DNA Methylation-Direct kit (Zymo Research).. Embryonic hearts at 48 hpf were dissected (n = 4 per condition).
| Cat # | Name | Size |
|---|---|---|
| D5001-1 | CT Conversion Reagent | 1 tube for 10 Conversions |
| D5001-4 | M-Wash Buffer | 6 ml |
| D5002-4 | M-Wash Buffer | 24 ml |
| D5001-5 | M-Desulphonation Buffer | 10 ml |
| D5002-5 | M-Desulphonation Buffer | 40 ml |
| D5001-6 | M-Elution Buffer | 1 ml |
| D5002-6 | M-Elution Buffer | 4 ml |
| D5005-2 | M-Dilution Buffer-Gold | 1.5 ml |
| D5006-2 | M-Dilution Buffer-Gold | 7 ml |
| D5005-3 | M-Binding Buffer | 30 ml |
| D5006-3 | M-Binding Buffer | 125 ml |
| D5020-7 | M-Solubilization Buffer | 4.5 ml |
| D5021-7 | M-Solubilization Buffer | 18 ml |
| D5020-8 | M-Reaction Buffer | 1 ml |
| D5021-8 | M-Reaction Buffer | 4ml |
| D5020-9 | M-Digestion Buffer (2X) | 4 ml |
| D5021-9 | M-Digestion Buffer (2X) | 15 ml |
| C1001-50 | Collection Tubes | 50 Pack |
| C1004-50 | Zymo-Spin IC Columns | 50 Pack |
- Catalog#: D5021 /D5020
- Package Length (in Inches): 9 /7
- Package Width (in Inches): 6.5 /5
- Package Height (in Inches): 6 /4.5
- Package Weight (in Pounds): 2.5 /0.5
- Size: 200 Rxns. /50 Rxns.
- Unit Standard: Metric
- Volume Units: Milliliters
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