EpiJET Bisulfite Conversion Kit

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/).