Hi,

from my experience it means that your primers are working but the probe consensus sequence is not present. You could sequence the PCR product and in case you know the probe sequence compare. Otherwise you might be able to ask the TF tech support afer obtaining the seqeuncing results if the probe will bind to the sequence found.

Apart form this if you want to know if the probe works you could run a sample where you know the probe should bind

Sven

To add: There could also be a problem with the machine's optics / detection if you can find the appropriate sized product when you run out the sample on a gel but the machine shows no amp.

Tamara Schenekar But how were the final concentration of the primer and probe? And how was the thermoprofile? And are you sure that your machine detect this type of probe? And you are sure for the quecer?

Hi,

as Tamara said that a similar assay with same modifications does work I would exclude instrument issues

Sven

Dear all, thank you very much for your answers, so far!

Sven Reister I have sequence information for my sample and the probe should match 100% with the probe sequence. I am running my qPCRs with tissue samples for now, so sample quality and template DNA quantity should also be no issue.

Craig Silver As mentioned, I did run a similar assay with this machine (a Rotor Gene) just a few weeks earlier which works fine. But I will set up a run that contains both assays in parallel, to be 100% sure.

Giorgia Pertile The final concentrations were 500 nM for primers and 300 nM for the probe. The thermoprofile is 95°C for 10 min and then (95°C for 30 sec and 60°C for 1 min)x 45 cycles. The other assay that is working fine uses the same fluorescence marker (FAM) and quencher (Q500), so this should be fine for this machine and settings.

Tamara Schenekar did you run a singleplex or multiplex? In never used the quecer Q500 and the FAM. Do you try with another different probe from FAM?

Giorgia Pertile I did also try with BHQ-1. Also, as I said, another assay using FAM & Q500 is working fine. So this combination seems to be working fine.

Tamara Schenekar but in this analysis you would like to amplify only one gene or to optimase a multiplex?

Tamara Schenekar another idea is to try to increase or decrease the temperature of annealing. For example, I try to analyse by TaqMan the 16S and in publication, I saw that it work at 57°C and not at 60°C.

Giorgia Pertile I am running only one primer pair, so a single plex. I just re-checked the machine with another primer pair/protocol, which worked fine, so the optics of the machine, as well as detecting exactly this fluorescence dye and quencher is also working.

I will try a lower annealing temperature, even though I am then worried about the specifity of the assay then. Because it is aimed to be run on environmental DNA samples in the future and should only amplify the target species out of the bulk DNA from those samples. But it would be good the know if that is the problem :)

Another idea is to try to prepare different mix and change the concentration of the probe. How much DNA did you use? Tamara Schenekar

Giorgia Pertile thank you, I will also vary the probe concentration. I used a genomic DNA extract from tissue with approx. 40 ng in the whole reaction. I read that this might be very high, so I will also perform a run with a dilution series of the template.

Another question that came up during my troubleshooting: Can the probe be too close to one of the primers? I've noticed that the probe is positioned only 3 bp away of the 3' end of the forward primer, could that be a problem, in the form that extention starts "too fast" before the probe can anneal properly (especially because I am using a two step protocol)?

Hi,

it is normally always the case that the Probe is in proximity to the Forward Primer to get a signal as soon as the probe has bound and to facilitate fast cycling. This should not cause issue as far as I know. Do you have the option to replace the probe? Guess it is a commercial available assay right? Did you reach out to the vendor with this question?

Sven

It's possible because I never see the site of the attached probe more near to the forward primer. In case is possible for to draw yourself the probe?

Dear Sven Reister & Giorgia Pertile the Primers + Probe came were published by another research team in the framework of another study. I will also contact the authors to hopefully get some advice from them. Re-designing the probe would be tricky (because the amplicon is a small fragment only, and you try to design a species-specific probe within that region, letting not much options to move it around), so probably the whole assay (so, probe AND primers) would need to be redesigned. I hoped to be able to avoid that, especially becaues it would mean re-ordernign the probe (which is quite expensive given the MGB, quencher and fluorescence labelling)