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Genomic DNA Isolation

How much genomic DNA is needed for bisulfite conversion using the MethylCode™ kit?
When using the MethylCode Bisulfite Conversion Kit, 500pg - 2 μg ng of genomic DNA is needed… For optimal conditions, use 200 – 500 ng.
NOTE: For GC-rich regions, sample amounts greater than 500 ng may result in incomplete bisulfite conversion.

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 available from Invitrogen for the isolation of genomic DNA.

Can I use formalin-fixed DNA for bisulfite conversion?
This is usually not very successful.  In general, cross-linked or damaged DNA is a poor starting material.

Does my sample need to be free of RNA for bisulfite conversion?
No, residual RNA is not going to interfere with the conversion reaction.

Does enzyme digestion before bisulfite conversion help mitigate imcomplete conversion?
When studying multiple genes, due care when using a restriction enzyme to avoid cut within a target region. 

Bisulfite Conversion

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.

How long can I store the CT Conversion Reagent in the MethylCode™ Bisulfite Conversion Kit after it has been dissolved?
The CT Conversion Reagent can be stored for one day at room temperature, one week at 4 C, one month at -20 C, and for up to 6 months at -80 C without a noticeable loss in quality.

The protocol describes dissolving the CT Conversion Reagent in a total of 1.25 mL, and it indicates that it should be sufficient for 10 reactions.  However, the protocol later mentions that 130 μ L is required for each reaction, which adds up to 1.3 mL.  Is this a mistake in the protocol?
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.

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.

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 C, and two to four months at -20 C.  We recommend on storing your converted DNA below -70 C whenever possible.

NOTE: The DNA is single-stranded and inherently less stable than dsDNA.

How can I quantify my DNA after bisulfite conversion?
Checking your sample by O.D.260/280 may be inconsistent for reasons that we’re still investigating.  We suggest performing qPCR or agarose gel electrophoresis and comparing your sample to a known amount of DNA.

Methylation-specific PCR*

How much of the bisulfite converted DNA should I use in PCR?
Use 2 - 4 μL of elution for each PCR reaction.  This translates to a minimum of 100-200 pg of starting DNA per reaction (quantified before conversion).

What is a maximum base pair length recommended for amplifying bisulfite modified 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.

My PCR machine can only be set to 100 μ L, but the MethylCode™ Bisulfite Conversion protocol recommends using 150 μ L in the conversion reaction.  Is this a mistake?
We have found that 150 μL works fine for the conversion reaction with most PCR machines.

What PCR settings are optimal for amplifying bisulfite modified DNA?
Optimal PCR conditions vary. We recommend starting at 40-45 cycles and using a hot start taq polymerase such as Platinum® Taq DNA Polymerase.

My PCR post-bisulfite conversion failed.  What can you suggest?
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 Hi Fidelity or AccuPrime™ Taq DNA Polymerase.

DNA:
Ensure that total template DNA is less than 500 ng.

I think my DNA may be under-converted (incomplete).  What do you recommend?
Ensure that the DNA used for bisulfite conversion is pure (see related FAQs regarding DNA source/quality).  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 

MethylCode™ Bisulfite Conversion Services

What is the best way to ship samples to minimize nucleic acid degradation?
Samples should be shipped on dry ice.

* NOTE: Methylation specific PCR may be covered by one or more of U.S. Patents Nos. 5,786,146, 6,017,704, 6,200,756 and 6,265,171 and patents based upon foreign counter-part applications.  No license or rights under these patents to perform methylation specific PCR is conveyed expressly or by implication to the purchaser of this product. User’s of Invitrogen Corporation’s products subject to this label license should determine whether they have all the appropriate licenses in place. Further, no warranty is provided that the use of these products will not infringe on the patents referred to above.