C3 on probe will not stop primer extension, it will only stop the probe to act as primer

Péter Gyarmati the proccess has been working in another lab , and the spacer blocks amplification there.

The C3 spacer is commonly used as a modification at the 3' end of oligonucleotides to prevent further extension during PCR amplification. It acts as a steric hindrance by introducing a bulky spacer between the primer and the template, preventing the polymerase from extending the primer further.

However, it is important to note that the efficiency of the C3 spacer in inhibiting amplification can vary depending on the specific experimental conditions and the characteristics of the polymerase being used. While the C3 spacer is generally effective at reducing amplification, it may not completely block extension in all cases.

There could be several reasons why you are not observing a clear difference in band sharpness between the LNA probe with the C3 spacer and the one without:

It is recommended to consult the literature or technical resources specific to the LNA probes and the polymerase you are using to understand their compatibility and optimize the experimental conditions for effective amplification inhibition. Additionally, you may consider alternative strategies for inhibiting amplification, such as using modified nucleotides or other blocking agents, depending on your specific experimental requirements.

Regenerate response

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Nikolay Klyashtornyy very useful information. Thank you.