DNA Methylation Analysis Support—Troubleshooting

When there is very little or no methylated DNA, MBD protein also binds non-methylated DNA to some extent. So it is important to follow the right protocol when using low DNA input. The product manual has different protocols specified for different DNA input amounts. 

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.

For next-generation sequencing troubleshooting help, please visit the troubleshooting pages within our Next-Generation Sequencing Support Center.

Please go here for troubleshooting help.

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.

For the 7500 Fast Real-Time PCR System, if the software version is below v2.0.4, the compatible HRM software is v2.0.1. If the 7500 Fast Real-Time PCR System has been upgraded to software version 2.0.4 or above, then you will need to get HRM Software version 3.0.1. 

There are a few possibilities: 

• First, ensure that the HRM Software version is v2.0.1, and the 7900HT Fast Real-Time PCR System software version is v2.3 or above. 
• Second, check that the run method used was as recommended in the HRM protocol; make sure the ramp rate for the dissociation stage is 1%. 
• Then try to open the calibration file from the HRM software, if it does not open, then the calibration file is defective. The defects could due to a bad calibration plate or instrument uniformity issue.