Hi,

I see intense smearing in the lanes with template. Smearing might be a sign of two much template but you don't seem to have to much template - however I would consider 45 cycles as far to much. This could also result in smearing.

So reduce your cycle mumber (i would not recommend more than 35 cycles) and you can try using more template DNA (I am not recommending more than 100 ng of genomic DNA).

I would also consider your extension time as to long - for a Taq polymerase for amplicons of your size 30 sec is more than enough (and prolonged extension times can have a negative effect on your PCR results).

Try to add DMSO (3-10%) (or BSA) to the PCR - this can enhance specificity.

Do a hot start - if you don't have a hot start polymerase simple start the PCR machine and keep the sample on ice - put them in the PCR machine when it reaches 95°C and close the lit).

Your MgCl concentration might be also an issue - this can vary from assay to assay

What I am missing is a positive control for the PCR you are showing - so do a positive control for every template and PCR you are running. Even if other PCR have worked on the template before, template can get degraded and if you don't include a positive control every time you don't know if your template is still intact.

Are you sure that the primers you are using a still ok? Sometimes primers can degrade as well - however as this seems to be a new PCR for you I woulld think that you have "fresh" primers and this is most likely not the reason for PCR failure

I would also include a PCR control which would dedect DNA which is not bisulfite converted (maybe for your lambda DNA or a DNA sequence outsite a CpG island)

What is not clear to me how you use or lambda- DNA for control. Do you spike your template before bisulfite conversion with the lambda DNA? This is the way it should be done. However how do you subsequently analyse the succsessful bisulfite conversion? Do you have special primer for this? Do you sequence the lambda DNA?

There is a lot you can try - this is what I would do first:

1: check you template integrity with a positive control (PCR that worked befoe)

2. Add DMSO

3. reduce your cycle number and your eytension time

4. Do a hot start

5. Try more template if the template is fine but don't use to much of the template - I would suggest 25 ng, 50 ng and 1000 ng (with reduced cycle number)

If nothing is working consider new primers

I hope this helps

Hi

It looks like you’ve done a solid job ruling out most obvious issues (like template quality, primer dimers, and bisulfite conversion efficiency), and I know bisulfite PCR can be notoriously tricky because of the DNA degradation and C→U conversion. A few things you might try: