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查看更多产品信息 MethylCode™ Bisulfite Conversion Kit - FAQs (MECOV50)
21 个常见问题解答
Ensure that the DNA that you use with the MethylCode Bisulfite Conversion Kit is ultra clean. 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, prior to performing the conversion reaction
Checking your converted sample by O.D.260/280 tends to be inconsistent. We suggest performing qPCR or agarose gel electrophoresis and comparing your sample to a known amount of DNA to quantify your DNA after bisulfite conversion.
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 on storing your converted DNA below -70 degrees C whenever possible (remember: the DNA is single-stranded and inherently less stable than dsDNA).
The CT Conversion Reagent that has been prepared in solution may be stored up to 1 week at -20 degrees C. We recommend thawing at room temperature and mixing for 2 mins by rotating or vortexing before use.
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/).
We have found that 150 µL works fine for the conversion reaction with most PCR machines.
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).
Yield of DNA after bisulfite conversion (when input of DNA is 200-500 ng) is ~70-80%. We suggest performing qPCR or agarose gel electrophoresis and comparing your sample to a known amount of DNA. Since the bisulfite converted DNA strands are no longer complementary, this DNA mainly stays in a single stranded form and resembles RNA. So if spectrophotometer method is used, converted DNA concentration needs to be calculated using 40 µg/mL for absorbance at 260 nm = 1.0.
The powdered chemicals will add to the total volume of the final solution. The volume should be greater than 1.4 mL after completely resuspending the CT Conversion Reagent as indicated in the protocol.
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.
When using the MethylCode Bisulfite Conversion Kit, 500 pg-2 µg of genomic DNA is needed. For optimal conditions, use 200-500 ng. Please note that for GC-rich regions, sample amounts greater than 500 ng may result in incomplete bisulfite conversion.