This experiment describes the process of how to customize a 40bp oligonucleotide. This experiment was derived from PCR Lab Guide (Second Edition) by Seed Kang and Qu Lijia.
Operation method
Customized 40bp oligonucleotide experiments Move 1. Make a top strand The TRAFIG program can be run with no genes to translate, but the output can be specified as a linear strand of 40bp length and degree. This conveniently provides a list of the justice strands of the designed sequence, ordered by the 40bp linear strand. Each linear chain can then be labeled with a name or number, and thus easily customized by E-mail from the vendor. The last segment of less than 40bp is not available for ordering. For more product details, please visit Aladdin Scientific website.
2. produce antisense chains The OTHSTR program was used to generate complementary chains, since both chains were customizable for assembly. Before running the TRAF1 G program to generate antisense strands of 40bp each, it is important to note (with the small number of bases at the beginning deleted) that the output should start at exactly the 20thbp on the right side of the justified strand mapping (the customized first segment of the linear strand should terminate at exactly the 21st nucleotide of the last oligonucleotide segment of the justified strand). The goal is to establish a precise 20:20 overlap in the assembly of the entire gene, as described by Stemmer et al. (1995). If the resulting map starts on the right, then the last linear strand is only 20 bases. This 20bp oligonucleotide is also not customized.
3. Customize a "touch-up" oligonucleotide. It is possible that the desired gene is not exactly the right length (40n+20), so an additional compensatory 40bp oligonucleotide needs to be customized to replace some of the bases that were missed at the start of the antisense alignment, which is a small number of base pairs beyond the gene. This additional oligonucleotide is more than 20 bases more than the right side of the antisense strand, but the "touch-up" oligonucleotide will serve as a template to participate in the assembly process and extend the gene by the small number of base pairs necessary to extend the gene beyond the gene because of the homology of these bases to the PCR primers on that side (see Figure 33-4). 33-4). In fact, as long as the overlapping region is at least 20 bp, "touch-up" oligonucleotides can be made on either side (the sense strand on the left and the antisense strand on the right) to change or adjust the edges of the desired artificial gene. 
4. Standard length (~25bp) PCR primers can be customized to guide the amplification reaction. Alternatively, depending on the cloning strategy employed, the first and last 40bp oligonucleotides can be used directly as PCR primers if they are used as full length in the reaction (200nmol/L).