On the other hand, when we digest the plasmid that possesses the optimized sequence of codons for the ovoalbumin protein with the cutting enzymes BamHI and PsiI, the result reported in column 2 of Figure \ref{272503} is obtained.
3.3.2. Linking the gene to be cloned and the plasmid
With the above digestions, the next step is to perform the ligation between the gene to be cloned and the plasmid, to perform this ligation and run it in the same gel we are located in the plasmid that has inserts the optimization of codons and we digest this with the enzyme of restriction PsiI that cuts only once (this is done because the plasmid has a circular shape, but when cut with the enzyme the plasmid opens up linear). Reporting in column 3 of Figure \ref{272503}.
In this way the plasmids to be cloned are built and the insertion of the ovalbumin protein is validated correctly in the plasmid.
3.3.3. Polimerase chain reaction
To perform the PCR process is part of the design of a Primer in the region before the gene to be cloned and a Primer located within the same gene (these Primers must have a melting temperature around 58°C and an approximate size of between 18 to 25 base pairs for each). The Primer located in the forward position has 24 base pairs with a melting temperature of 58.00°C and the other Primer in reverse orientation consists of 24 base pairs with a melting temperature of 58.37°C. Subsequently, the two Primers are linked to the product to obtain PCR, obtaining 412 pairs of bases that can be seen in Figure \ref{815030}.