How can I test that my primers pair design is correct?

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Satyendra Mondal

Hello Abdu Alotaibi

It seems that your forward primer cannot be found in the template. The forward primer should be identical with the template sequence, not complementary.

If you are willing to perform regular PCR-based amplification, use primer-BLAST or similar primer-designing software. Copy-paste your experimental DNA sequence in the in the template box. Define parameters like the region of forward and reverse primer in the sequence, the length of the amplicon, the melting temperature, it will design primer for you.

Best regards.

Katie A S Burnette

Looks like you are using Primer-BLAST, which is a great tool.

If I'm understanding correctly, you want to use an already-designed left primer. Reverse complement the DNA sequence in the text box and I bet it will work.

Good luck!