I used a KOD DNA polymerase for PCR that works very well. Then I used this enzyme to run a TaqMan probe real-time PCR but the results waere unexpected. I attached the results. The fluorescence rise very fast during initial cycles and it reach to a plateau much faster than the control. The control enzyme is a Hot-start but the KOD is not. Can this be the cause of this phenomenon or is there another reason involved?
figure legend:
Red: Control enzyme (Hot-start)
Blue, green, purple, and pink: KOD with different buffer.
Have you analyzed the reaction on an agarose gel? I'd expect to see a lot of unspecific products. If not, there might be too much activity of the KOD polymerase.
Hi dear,
It is look like that you have to adjust the concentrations. Pls use different concentrations with different primer's amount and plot them to decide about the most suitable one for your stuff.
Start with 1 ng template and go gradually through 6 concentrations up to 10 ng. Performe this with different primer's amount such 0.3 and 0.6. I think this will give you a hand.
Best...
Hi
Yes, I also agree you need to optimize the reactions first, what ever polymerase you are using. Also the nature of enzyme (hot start/regular) will also make difference.
Hi..
There are several potential reasons why you may have observed unexpected results when using KOD DNA polymerase for TaqMan probe real-time PCR.
One possibility is that the KOD DNA polymerase has a higher intrinsic activity than the control enzyme, leading to faster amplification and fluorescence rise during the early cycles of the PCR. The fact that the KOD polymerase is not a hot-start enzyme could also contribute to the faster amplification, as hot-start enzymes are designed to inhibit polymerase activity at lower temperatures, which can help to reduce nonspecific amplification and improve the specificity of the PCR.
Another potential reason for the unexpected results could be the composition of the buffer used with the KOD polymerase. Different buffers can affect the activity and stability of the polymerase, and may also affect the efficiency of the PCR reaction. It is possible that the buffers used with the KOD polymerase are not optimized for TaqMan probe real-time PCR, leading to the observed differences in fluorescence rise.
To determine the cause of the unexpected results, you may want to try repeating the experiment using different concentrations of the KOD polymerase and different buffer conditions to see if these factors have an effect on the PCR performance. You could also consider using a different real-time PCR platform or assay to confirm the results.
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