In my lab we carried out qRT PCR for checking the efficiency of the primers. The results came fine for dilutions, unfortunately for non template control also we got good expressions. Inorder to find out what may be the possible reason we did Agarose Gel Electrophoresis and unfortunately we did not get any amplification. Also repeatedly checked with fresh same set of primers and we got Ct values. What might be the reasons for this to happen? Do we have to doubt the Primer designing process? Is there will be any difference if we use SYBR green with ROX and without ROX?
It would be helpful to know the actual Ct values. If these are rather high (>35), it's likely that primer-dimers are amplified. They can be hard to see in a gel, because they are very short and their length may be diffuse (better use PA gels!). Also check the melting curves. These prducts should have a regognizeably lower Tm than your specific products.
Can you share an image of the curves? Can you share an image of the gel and what percent agarose and the animated length of the amplicon? The most proble explanation is contamination of one of your reaction components, which can easily happen if you are using pipet tips that are not filtered. Sometimes as well the primers will form dimers and create CT signal that may not appear on the agaorse gel as a discrete band.
It would be helpful to know the actual Ct values. If these are rather high (>35), it's likely that primer-dimers are amplified. They can be hard to see in a gel, because they are very short and their length may be diffuse (better use PA gels!). Also check the melting curves. These prducts should have a regognizeably lower Tm than your specific products.
A contamination could be the reason (maybe in buffers or oligo preparations).
Andreas Eisenreich , in this case one would get a band in the agarose gel, what is not observed.
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