This year, our project designed a new part BBa_K4801000 from uncultured Bacillus sp. We connected it to pET-28a and imported it into E.coli (DE3) to obtain nitrite reductase, which was loaded onto an electrode for the degradation of food nitrite. Here are some design experiences we would like to share:

1. Primer length: Textbooks require 15-30bp, commonly around 20bp. In reality, it is best to use 18-24bp to ensure specificity, not as long as possible. Primers that are too long will also reduce specificity and yield.

2. Primer amplification span: It is appropriate to use 200-500bp, and under specific conditions, it can expand to 10kb fragments.

3. Primer base: The appropriate content of G+C is 40-60%. If there is too little G+C, the amplification effect is not good. If there is too much G+C, non-specific bands may appear. It is best to randomly distribute ATGC to avoid a string arrangement of more than 5 purine or pyrimidine nucleotides. The 5 'end and intermediate sequences should have more GC to increase stability and avoid GC rich at the 3' end. The last 3 bases should not have GC, or the last 5 bases should have 3 without GC.

4. Avoid secondary structures within primers, and avoid complementation between two primers, especially at the 3 'end. Otherwise, primer dimers will be formed, resulting in non-specific amplification bands.

5. The base at the 3 'end of the primer, especially the last and second to last base, should be strictly paired to avoid PCR failure due to non pairing of the end base.

6. If there are or can be appropriate cleavage sites added to the primers, it is best for the amplified target sequence to have appropriate cleavage sites, which is very beneficial for cleavage analysis or molecular cloning.

7. Primer specificity: Primers should have no significant homology with other sequences in the nucleic acid sequence database.