can anybody suggest why qPCR reaction dose not work although when I load the qpcr product on gel, it appears positive, there is clear band on gel electrophoresis.
There are a lot of factors in this situation. One factor is the salinity conditions when you performance the PCR and when you performance the qPCR. I'll try to performance both PCR in the same conditions (same buffer, same DNA quantity, etc.).
Another factor is that the PCR protocols must have the same temperatures with the same ramps. There are small differences in tenths of a degree when you performance the PCR and qPCR.
Maybe, your reactions begin to amplify in the last cycles and you should to add more cycles in your qPCR protocol.
I hope I've help you. Regards.
Hello Eljelani,
If I understood correctly, you are unable to see amplification on the qPCR machine but can see bands when the same reaction mixture is run on a gel. If there is an amplification, it means that the reaction mixture and temperature cycles for suitable for amplification (just to clarify....you are sure those bands are not primer dimers??). As for not being able to see the amplification on the Ct curve on the system, it is possible as Alejandro mentioned for that amplification to be occurring in the later cycles, so maybe try to increases the number of cycles and see if you can catch the amplification.
Also, please check the software settings, just to check if the machine is auto-normalizing all results to the blanks (wells specified as negative controls) and also at the melting curve...if there is an amplicon/amplicons you will see it for sure via the melting curve. Mainly, just to identify whether the error is on the end of the machine or the reaction set-up. Hope this helps.
Regards
Hi,
It could be also possible that you see primer dimer in your gel. Maybe you should make a 2% Agarose gel and let it run for a long time, so that you can see the differences in the size of you positive control and the sample. In General the qPCR-fragments are very small. You can also try to clone this Fragment in a pCR4 Topo vector and sequence. And a melting curve also helps.
thanks all guys for advice and suggestions. Regarding primer dimer, it is easy to differentiate it from my expected band, 161bp, on 2% agaros running for 1:25 hour especially when running on 100bp ladder. in addtition the qpcr product was sequenced to confirm the species. I think that there is some problem with my prob but I can point it: series anaeling and concentration were don but unfortunately, there was still no amplification on qPCR machine.
thanks again for help and support.
you are correct Alejandro_Mendoza and Aparajita Lahree. I have seen very late raising in the curve although the cycle number that was used 45 cycles. I am not sure if it worthwhile increasing it more, 50 or 55 for instance.
Possibly: your primers work (and therefore amplify the DNA), your probe does not bind the amplicon, so it cannot be detected in the qPCR ongoing reaction, but can be using other methods. If so, try another probe, check probe sequence, check the qpcr machine is set to correctly detect the flourescent tag on the probe, check there are no funny sequential anomalies (snips, alternative splice sites and so on) around where you think the probe should bind.
You could sequence your amplicon to make sure it has a sequence your probe will detect.
Or just ditch the probe and use SYBR green.
thanks Robi, I will try working on those possibilities, and hopefully will work.
Regards,
- Badges
- Science method