I am running qPCR with gDNA. Using primers tested in a previous experiment, I am not seeing any exponential amplification. In fact, most samples (unknowns and my standards) return with an undetermined quantity and Ct.
The gDNA was nanodroped: 373.3ng/uL with a 260/280 of 1.88.
I have run this experiment several time previously with different samples and primers and have never had this issue.
Did you check in normal PCR , it is giving the amplification or not,.
Is primer old ? similar condition you are using for both PCR or different.
keep positive control also. and negative
I would recommend using a negative and positive control. If you are still encountering such an image;
Check primer activity. It may be annealing to other regions. How are your melting curves? If you have more than one peak, it may be that the primer is annealing to other places. You can also increase the Mg concentration. And check the extension and annealing times. Before starting work, sterilize the environment and work on ice. Anneal at 5 or 4 ° C degrees below the Tm. Also pay attention to the high purity of the water you use. After DNA isolation, there may be some PCR inhibitors (eg phenol).
Best wishes,
Tuğçe
It looks to me that you got zero amplification of your DNA. Look at the scale on the Y-axis you'll notice that you have less than 1X change in delta Rn. The "jagged line" is an artifact of the fact that you have basically zero change, but it's been scaled to take up most of the screen.
Include a good positive control to control in your qPCR and you won't see this sort of graph.
With all the above suggestions. Your gDNA concentration is very high. You need to dilute it with ultra pure water in order to minimize inhibitors.
I would recommend you:
1- Dilute your gDNA up to 50 ng/uL with the total reaction volume 10 uL.
2- Design and select the primers with the higher annealing temperature (60-62) that can help you to get a specific pics with 10ng/uL (10picomol).
3- You must get specific bands with a clear result on the gel performed using the conventional PCR, but you have to take care regards to the polymerase, the better is similar with the fluorescence.
4- Using housekeeping gene will be a good indicator for the experimental conditions.
5- Finally, run the qPCR experiment.
Note: the 260/280 ratio might be not less than 2.5, please check the nanodroped instructions.
It looks like that your primers are not working on this set of cDNA. Several possible reasons could be:
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