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Thermo Scientific™ EpiJET Bisulfite Conversion Kit

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FERK1461 50 reactions
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Catalog No. FERK1461 Supplier Thermo Scientific™ Supplier No. K1461
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Description

The EpiJET Bisulfite Conversion Kit is used for an efficient bisulfite modification of DNA for methylation analysis.

Thermo Scientific EpiJET Bisulfite Conversion Kit is designed for simple and reliable bisulfite conversion of DNA for methylation analysis. In the bisulfite reaction all unmethylated cytosines are deaminated and converted to uracils, while methylated cytosines remain unchanged. Bisulfite converted unmethylated cytosines are detected as thymines in the following PCR.

The EpiJET Bisulfite Conversion Kit integrates the thermal DNA denaturation and the bisulfite conversion reactions into one step. This step can be performed following long or short protocol, depending on the downstream application of modified DNA. The converted DNA is when bound to a membrane of a micro column for on-column desulfonation and subsequent DNA purification steps. The converted DNA is eluted in a small volume of Elution Buffer and is suitable for a number of techniques used for the methylation status analysis, including PCR, qPCR, COBRA, sequencing.

Highlights

  • High conversion efficiency and specificity (≥99%)
  • Flexibility, provided by short DNA conversion protocol
  • Pure DNA, suitable for downstream applications and long-term storage

Applications

Converted DNA can be used in:

  • PCR
  • qPCR
  • Combined Bisulfite Restriction Analysis (COBRA)
  • Sequencing

Includes

  • Modification Reagent
  • Modification Solution I
  • Modification Solution II
  • Binding Buffer for Bisulfite Conversion Kit
  • Wash buffer (conc.) for Bisulfite Conversion Kit
  • Desulfonation Buffer (conc.)
  • Elution Buffer
  • DNA Purification Micro Columns and Collection Tubes

Specifications

Quantity 50 reactions
Product Type Bisulfite Conversion Kit
Product Line EpiJET

Frequently Asked Questions (FAQs)

My PCR post-bisulfite conversion failed. What can you suggest?

Here are some recommendations:

- Primers: Ensure that your primers are designed to amplify the converted template. We recommend primers that are 24-32 nts in length and contain no more than 2-3 mixed bases (for base-pairing to C or T residues). The 3' end of your primer should not contain a mixed base or end in a residue whose conversion state is unknown.
- Polymerase: We recommend using a hot-start Taq polymerase such as Platinum Taq DNA Polymerase, Platinum Taq High Fidelity, or AccuPrime Taq DNA Polymerase. Proof-reading polymerases are not recommended because they cannot read through uracil present in DNA templates.
- Amplicon size: Bisulfite modification is harsh and may cause strand breaks. Most research publications recommend 200 bp lengths. Larger amplicons can be generated, but this requires an optimized protocol.
- Template DNA: We recommend using 2-4 µl of eluted DNA for each PCR reaction. Ensure that total template DNA is less than 500 ng.
Where can I find troubleshooting tips for Sanger sequencing of my bisulfite converted DNA?

Please use the following link (http://www.thermofisher.com/us/en/home/technical-resources/technical-reference-library/capillary-electrophoresis-applications-support-center/sanger-sequencing-support/sanger-sequencing-support-troubleshooting.html) for troubleshooting help.
Where can I find troubleshooting tips for next-generation sequencing of my bisulfite converted DNA?

For next-generation sequencing troubleshooting help, please visit the troubleshooting pages within our Next-Generation Sequencing Support Center (thermofisher.com/ngssupport).
I think that my DNA may be under-bisulfite converted (incomplete). What do you recommend?

Ensure that the DNA used for bisulfite conversion is pure. If particulate matter is present after adding the CT Conversion Reagent, then centrifuge the material at high speed and perform the conversion with the clear supernatant. Also, ensure that all of the liquid is at the bottom and not in the cap or walls of the PCR tube before performing the conversion reaction.
What program can I use to design primers for methylation mapping experiments?

You can use Methyl Primer Express Software to design primers for methylation studies. You can download the program for free from here (http://resource.thermofisher.com/page/WE28396_1/).
What is a maximum base pair length recommended for amplifying bisulfite converted DNA?

Bisulfite modification is harsh and may cause strand breaks. Most research publications recommend 200 base pair lengths; however, larger fragments have been amplified with an optimized protocol.
Can bisulfite converted DNA be used for Sanger sequencing?

Yes. For more information, please go here (http://www.thermofisher.com/us/en/home/technical-resources/technical-reference-library/capillary-electrophoresis-applications-support-center/sanger-sequencing-support.html).
Can bisulfite-converted DNA be used for next-generation sequencing?

Yes. We have tried using the EpiJET Bisulfite Conversion Kit with next-generation sequencing, and it works really well (conversion of unmethylated Cs was >99.98% when using Protocol A; the 30 min protocol is not recommended for next-generation sequencing). For next-generation sequencing support, visit our Next-Generation Sequencing Support Center (thermofisher.com/ngssupport).
What is the difference between Protocol A and B in the EpiJET Bisulfite Conversion Kit?

-Protocol A results in DNA conversion efficiency >99%, is more sensitive and gives lower DNA degradation levels than Protocol B. This protocol is recommended for all routine epigenetic applications.
-Protocol B results in DNA conversion efficiency >95% and is much faster than Protocol A (30 min vs. 160 min for DNA denaturation/bisulfite conversion step). Modified DNA is suitable for PCR of amplicons up to 300 bp. This protocol is not recommended for small DNA amounts (less than 50 ng of input DNA). It is the best for fast DNA methylation screening.

What should I use to isolate DNA for bisulfite conversion?

The best conversion results are obtained with highly pure DNA. Most commercially available kits yielding good quality DNA can be used. The PureLink Genomic DNA Purification Kit is a complete kit we offer for the isolation of genomic DNA.
How should I store my bisulfite-converted DNA after it is eluted from the column?

The converted DNA is stable for one day at room temperature, one week at 4 degrees C, and two to four months at -20 degrees C. We recommend storing your converted DNA below -70 degrees C whenever possible.
Note: The DNA is single-stranded and inherently less stable than dsDNA.
Can I elute the bisulfite converted DNA with more than 10 µL?

Yes. There is no upper limit to the volume that can used to elute. If there is insufficient elution buffer remaining, then water or TE can be used to elute.
My DNA is highly supercoiled. Will that interfere with bisulfite conversion?

Yes, bisulfite conversion requires DNA denaturation. Supercoiled DNA (usually of plasmid origin) is typically more difficult to denature, which may lead to potential under-conversion.
Does my sample need to be free of RNA for bisulfite conversion?

No, residual RNA does not interfere with the bisulfite conversion reaction.
Can I use formalin-fixed DNA for bisulfite conversion?

This is usually not very successful. In general, crosslinked or damaged DNA is poor starting material.
How much genomic DNA is needed when using the EpiJET Bisulfite Conversion Kit?

Starting DNA amounts from 50 pg up to 2 µg can be used for DNA conversion using the EpiJET Bisulfite Conversion Kit. Nevertheless, for optimal results, use 200 - 500 ng of input DNA. High input DNA amounts may result in incomplete bisulfite conversion for some GC-rich regions.

For Research Use Only. Not for use in diagnostic procedures.