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 / 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.
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The integrated genomic landscape of thymic epithelial tumors
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…
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 …
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).
|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|
|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