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View additional product information for EpiJET Bisulfite Conversion Kit - FAQs (K1461)
16 product FAQs found
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.
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.
For next-generation sequencing troubleshooting help, please visit the troubleshooting pages within our Next-Generation Sequencing Support Center (thermofisher.com/ngssupport).
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.
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/).
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.
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).
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).
-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.
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.
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.
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.
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.
No, residual RNA does not interfere with the bisulfite conversion reaction.
This is usually not very successful. In general, crosslinked or damaged DNA is poor starting material.
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.