Hi Saikal,

there are a lot of factors that can cause failed ddPCR runs, ranging from the gDNA to PCR reagent and program.

1. Make sure that your extracted gDNA is of high quality by measuring its concentration and purity.

2. Following bisulphite conversion, make sure you measure the concentration again (e.g. using Qubit single stranded DNA), to make sure that your recover your input DNA.

3. Prior to running in ddPCR machine, it doesn't hurt to amplify your bisulphite-converted DNA using the primers on the thermocycler (without probe) and run the PCR product on a gel. A gradient annealing temperature can be useful too. This will help you decide if:

a. there's anything wrong with the PCR program,

b. your bisulphite converted DNA is PCR amplifiable,

c. your primers are diluted properly,

d. you are amplifying your target amplicon

4. Run the ddPCR program, including the probe this time, to see if the probe is working well.

A gradient PCR may or may not be necessary because you are using a previously established protocol. My suggestion is to wait until you have controls prior to troubleshooting and doing your experiment. Without controls, the experiment is uninterpretable and, though possible, difficult to troubleshoot. It is not worth the time and resources.

Good luck!

Dear Danny,

thank you so much for your kind reply and recommendations! this helps a lot! I will try all suggestions. Very appreciated for your time and answer. have a lovely day, Danny Laurent