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New Standard for DNA Methylation Analysis!
EpiSight Bisulfite Conversion Kit
EpiSight BisulTaq DNA Polymerase

EpiSight Bisulfite Conversion Kit (Wako Cat. No. 297-71901) offers a fast and efficient alternative to traditional bisulfite conversion. The protocol is optimized to minimize reagent preparation and bisulfite conversion time. This kit contains Enhancer for bisulfite conversion to allow the enhancement of the bisulfite conversion efficiency and inhibit of DNA degradation. Subsequent PCR-amplification using EpiSight BisulTaq DNA Polymerase*1, recombinant, Solution (Wako Cat. No. 297-72001) is more efficient compared to using conventional Taq DNA polymerase.

*1: Hot-Start Gene Taq NT (Nippon Gene)(Wako Cat. #311-07523 (250 units) is also ecommended,
      depending on DNA samples.

  INDEX

  1. Features
  2. EpiSight Outline
  3. Kit Contents
  4. Protocol Flowchar of Bisullfite Reaction
  5. Comparison of Bisulfite Conversion Efficiency using mononucleoside (HPLC)
  6. DNA methylation analysis of Fgf 4 promoter region in mES and M1 cell
  7. FAQ
  8. Product List

  9. Links ⇒ Leaflet
                   



1. Features

  • Minimizing DNA damage
  • High conversion rate of Cytosine to Uracil
  • Adoption of Wako's unique Enhancer
  • Efficient PCR amplification of bisulfite treated-DNA

2. EpiSight Outline


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3. Kit Contents

These kits do not include reagents for DNA and sequencing.


Additional materials required
  • Isopropanol (Wako Cat. No. 166-21671 (100 mL))
  • GelRed™ Nucleic Acid Gel Stain, 10,000x in Water (Biotium, Inc.)
  • GelRed™ Nucleic Acid Gel Stain, 10,000x in DMSO (Biotium, Inc.)
  • 6x Loading Buffer Triple Dye (Wako Cat. No. 314-90261 (1 mL x 3))
  • NaOH (97.0+%) (for Molecular Biology; Wako Cat. No. 190-14565 (500 g))
  • Ethanol (99.5) (Wako Cat. No. 052-07221 (100 mL))
  • Sterilized water (Wako Cat. No. 316-90101 (100 mL))
  • Ligation Reagent
  • dNTP mixture (2.5 mmol/each)
  • 0.2 mL PCR tubes
  • 1.5 mL tubes
  • Vortex mixer
  • PCR thermal cycler
  • Microcentrifuge (Capable of 14,000 rpm and 18,800 x g)
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4. Protocol Flowchart [Standard Protocol]






*2





The condition should be optimized depending on cell line, length of the template DNA and GC content.
In case that the template DNA is more than 500 bp, follow the above mentioned standard protocol.
In case that you got insufficient amplified products or insufficient conversion from cytosine to uracil,
incubate at 95℃ for 1 min. and then 60℃ for 30 min. for 32 cycles.
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5. Comparison of Bisulfite Conversion Efficiency using mononucleoside (HPLC)


Enhancer provided in EpiSight Bisulfite Conversion Kit achieved the ideal conversion reaction such as enhancement of C→U conversion reaction and inhibition of mC→T conversion reaction.
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6. DNA methylation analysis of Fgf 4 promoter region in mES and M1 cell



















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7. FAQ

Q: What is DNA methylation?
A: In case of eucaryotes, it is methylation of carbon at the 5 position of cytosine (C). The transcription factor cannot bind to methylated DNA region and gene expression is inactivated.

Q: What is Bisulfite Reaction?
A: It is a reaction in which cytosine (C) is converted to uracil (U) by sodium bisulfite. As methylated cytosine is not converted to uracil (U), methylation of DNA is analyzed utilizing this difference.

Q: What do ○ and ● in data show?
A: ○ shows thymine (T) and ● shows cytosine (C). In other words, ○ is the region of non-methylated cytosine and ● is that of methylated cytosine before bisulfite reaction. The line number presents each plasmid clone and the row number presents the cytosine (C) number designated in advance.
Q: What is the most notable feature of EpiSight Bisulfite Conversion Kit?
A: It is high conversion rate of cytosine (C) to uracil (U).

Q: What is your recommendable kit to prepare genomic DNA?
A: As extraction kits of genome DNA,
QuickGene SP kit DNA tissue (96 tests; Wako Cat. No. 632-23629)*3, QuickGene DNA tissue kit S (96 tests; Wako Cat. No. 637-23559)*3.
In addition, DNA extraction kits using general-purpose columns.
*3: It is available in Europe, only.

Q: Why is Taq DNA Polymerase sold separately?
A: Because there is a patent.

Q: Why is the reaction time shortened?
A: Because Enhancer promotes the Bisulfite reaction.

Q: Is it possible that the reaction time is shorter than 2 hours?
A: The shortest reaction time is around 90 minutes although It depends on the genomic DNA.

Q: I worry about the reaction at 95°C. Do you have any other reaction under a mild condition?
A: We confirmed the Bisulfite reaction at 55°C for 16 hours reduced the conversion efficiency.

Q: Does the high-temperature reaction have negative effects for my samples?
A: Please refer to the above mentioned "Q: I worry about the reaction at 95°C. Do you have any other reaction under a mild condition?".

Q: What is Enhancer?
A: The composition is proprietary. It is a catalyst used in the Bisulfite reaction.

Q: Is it OK to react other competitor’s kits at such high-temperature?
A: Follow the protocol of the kit.

Q: Can any Taq DNA Polymerase be used?
A: Use EpiSight BisulTaq DNA Polymerase (Wako Cat. No. 297-72001). It is the DNA polymerase optimized to PCR after bisulfite reaction. Use of α-type DNA polymerase is not recommended.

Q: Can I keep the bisulfite treated DNA sample?
A: You can keep it below -20°C for 2 weeks. However, the bisulfite-treated genomic DNA is unstable. Please use up the samples as soon as possible.

Q: What does 5-hydroxymethyl cytosine (5-hmC) become after the reaction?
A: It is not converted to uracil similarly to 5-mC. This kit cannot distinguish 5-hmC and 5-mC.

Q: Can the DNA samples treated with this kit be used in the next generation sequencing?
A: Yes, they can.

Q: Can a column be used as the purification of the bisulfite treated DNA instead of DNA Cleaner?
A: Yes, you can purify it using an appropriate column.

Q: What is the recommendable control DNA?
A: Totally methylated DNAs (e.g.: mouse M1 cell-derived DNA) and non-methylated DNAs (e.g.: mouse ES cell-derived DNA, cloned plasmid DNA) are recommended as controls to confirm proper conduct of bisulfite reaction.

Q: To what points should we pay attention to primer designing?
A: Pay your attention to the following points:
  1. Design forward primers from the sequence converted from cytosine (C) to thymine (T).
  2. Design taking care to prevent bias in base sequence at Tm of 62°C and higher.
  3. Design the PCR products which is 400 bp or less.
    Tm: Melting temperature at which a half of double-stranded DNAs divided into single-stranded DNAs.

Q: How many conversion kits do 100 units of EpiSight BisulTaq DNA Polymerase (Wako Cat. No. 297-72001) correspond to?
A: Roughly 2 ~ 4 kits depending on the method of use.

Q: What is "DNA Cleaner", a kit content of EpiSight Bisulfite Conversion Kit ?
A: It is a polymer solution to remove low molecular DNA.

Q: To what length can DNA be amplified by PCR?
A: Assuming that cytosines in a template DNA are "completely converted", amplification to approximately 600 bp has been confirmed by PCR.




7. Product List
Product Name Grade Package Size Wako Cat. No.
EpiSight Bisulfite Conversion Kit for Genetic Research 20 tests 297-71901
EpiSight BisulTaq DNA Polymerase, recombinant, Solution*1 for Genetic Research 100units 297-72001
*1: Hot-Start Gene Taq NT (Nippon Gene) (Wako Catalog No.311-07523 (250 units) is also recommended, depending on DNA samples

QUOTATION REQUEST for EpiSight kits
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Note

  • Listed products are intended for laboratory research use only, and not to be used for drug, food or human.
  • Please visit our online catalog to search for other products from Wako: https://www.e-reagent.com
  • This information may contain products that cannot be exported to your country due to regulations.
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