I use the amplicon to be a positive control for a multiplex taqman realtime PCR assay. So I designed the amplicon and checked the sequence of amplicon, its sequence is OK for the primers and probes.
But when I realtime PCR, the signals are Ok on gDNA but not ok on the amplicons (~1000 bp). I don't know why.
Are you saying that the PCR works fine on your samples but not on your positive control? If so, it seems likely that you have some kind of inhibition going on. Also, 1000bp is very long for a real time PCR amplicon...
Dear Jill Muhling,
Yes, I mean that the realtime PCR works fine on my gDNA samples.
My amplicon is a sequence (1000bp) include 8 target sequence regions (each region also has around 100bp) as the regions on genome and my experiment is a multiplex realtime PCR. You mentioned some kind of inhibition, can you help me figure them out? I don't think it related with chemical inhibitors or competition of primers.
I'm sorry if my English is poor.
Hi Hoang,
The control reaction include all the 8 primer pairs? With the corresponding probes too?
If so, it could be too much, and one amplification could interfere with the other. Did you test just one primer pair? Are you sure the concentration of the control is OK?
Dear Ariel, each reaction include 2 primer pairs and I have 4 control reactions for 8 regions. I want to detect wild type and mutant allele of each region.
My problem is reaction with control amplicon not fine at few region but fine with gDNA (at the same region)
Ok. The first question is now clear. Only two pair. But still only one target molecule with two site. In the gDNA are the PCR targets also near (one PCR zone continuos to the other)? Or, ...do the gDNA have only one of them?
Are the two sites near or in different zones of the "amplicon"? How do you obtain the amplicon? Synthesis? Is a plasmid? Lineal or circular? Maybe you have an scheme or some link to expain it easier...
And still: Did you test just one primer pair? Are you sure the concentration of the control is OK? Too hight or too low concentration of the amplicom can be the problem.
Dear Ariel,
In one reaction, I used 2 pairs primer to amplify 2 regions which on one gene (I don't know how many bp is called "near" as you mentioned above) and used 4 specific probes to detect the status allele (wild type or mutant) of each region.
Two my "control amplicons" are lineal and were designed like 8 regions on that gene (gene size is 1600bp). One amplicon include wild type status of 8 regions; one amplicon include mutant status of 8 regions. Then in control reaction, I mixed two type of amplicons together to be template.
When I tested on gDNA, the status of 8 regions were detect fine. But when I tested on mixed amplicons or single amplicon, only one of 8 regions was not detected (I mean no Ct value) while others work fine. So I don't think about the concentration of the amplicon. (the concentration of amplicon I used ~ 10E3 copy/reac).
I also sequened the control amplicon and their sequences were Ok.
- Badges
- Science topic