DNA Assembly (Site Directed Mutagenesis)
Design PCR primers that i) anneal adjacent to the mutagenesis site and ii) have primer tails that include the substituted nucleotides for the mutagenesis site.
Carry out PCR as described by the DNA polymerase enzyme manufacturer. Plasmid DNA template should at 1 -10 ng mass in the PCR mix.
Purify amplified DNA by gel extraction by selecting band of correct size to remove primers and non-specifically amplified DNA.
Carry out template DNA digestion, DNA ends phosphorylation and ligation for vector cyclisation using the KLD kit from NEB by using 100ng of purified DNA. Incubate the reaction for 1 hour at room temperature.
Transform cells with 5 microliters (50ng) of the assembled DNA.