It is important that the primers critical synthesis reaction

It is important that the primers critical synthesis reaction are phosphorylated at 5\' end. Why? In the first transformation, it is critical that the host be mutS, but this is not essential at the second transformation. Why? Explain the need for the Ndel digest prior to the second transformation. How much of a purification of mutated plasmids resulted from this digest? When designing the primers for this type of mutagenesis what feature of them (when comparing them) is essential?

Solution

1.It is important that the primers in the synthesis reaction are phophorylated at their 5’ end. Why?

Phophorylated 5’ end of the primer helps in the ligation of PCR product into vector which further continues the gene cloning and gene expression.

2.In the first transformation, it is critical that the host be mutS, but this is not essential at second transformation. Why?

MutS help in the sequence identification during the recombination for the mismatch repairing. Strains lacks the MutS have found increased mutation frequencies. This is not essential for second time transformation because that is present in that sequence. Because recombinant formed during second transformation will have mismatch repair system.