When making a substitution to a plasmid using Q5 should I use methylated plasmid as my template? If I have a plasmid purchased from a vendor should I first transform it into E.coli?
Usually the plasmid supplied by most vendors will be methylated. You can confirm by looking at what host strain the plasmid was prepared in, most vendors will list this in the specifications or they would give you the answer if you ask. The relevant factor for methylation is that the plasmid should NOT be prepared in a dam- strain.
the usual strains of E.coli used to propagate plasmid DNA are dam+/ this methylation is required for Dpn1 to cleave the template DNA (and thus reduced the background of parental DNA). PCR products are not methylated (ecept if you add methylated dNTPs in the reaction). chemically synthesized DNAs are not methylated either (exept if you asked for introduction of special bases for the synthesis)