EZ-96 DNA Methylation-Direct MagPrep
D5045 / D5044
Highlights
- High throughput, complete bisulfite conversion of DNA directly from blood, tissue, cells, FFPE and LCM-derived samples
- Compatible with small sample inputs – as few as 10 cells or 50 pg of DNA.
- High throughput (96-well), automated desulphonation and recovery of bisulfite-treated DNA.
Description
| Applications | 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 | ≥ 25 µl |
| Equipment | Thermocycler with heated lid, heating element for 96-well plate, magnetic stand. |
| 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 |
| Recovery | > 80% |
| Sample Source | Blood, tissue, cells, FFPE, LCM-derived samples, purified genomic DNA, endonuclease-digested DNA, linearized plasmid DNA, etc. |
| Supplemental Info |
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 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 μg/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.
| Cat # | Name | Size | Price | |
|---|---|---|---|---|
| D4100-5-8 | MagBinding Beads | 8 mL | €92,00 | |
| D4100-5-16 | MagBinding Beads | 16 mL | €166,00 | |
| C2003 | Elution Plate | 2 Plates | €21,00 | |
| C2002 | Collection Plate | 2 Plates | €23,00 | |
| C2005 | Conversion Plate w/ Cover Foil | 2 Plates/Foils | €15,00 | |
| D5021-7 | M-Solubilization Buffer | 18 ml | €28,00 | |
| D5021-8 | M-Reaction Buffer | 4ml | €21,00 | |
| D5021-9 | M-Digestion Buffer (2X) | 15 ml | €15,00 | |
| D5040-3 | M-Binding Buffer | 250 ml | €105,00 | |
| D5040-5 | M-Desulphonation Buffer | 80 ml | €74,00 | |
| D5040-4 | M-Wash Buffer | 72 ml | €68,00 | |
| D5007-6 | M-Elution Buffer | 8 ml | €10,00 | |
| D5006-2 | M-Dilution Buffer-Gold | 7 ml | €10,00 | |
| D5003-1 | CT Conversion Reagent (96 Conversions) | 1 Bottle | €63,00 | |