Hello, I am reading the Q5 Site-Directed Mutagenesis (NEB) protocol. I understand that the phosphorylation step is needed for subsequent ligation. But I wonder what is the principle of removing the template plasmids by Dpnl. Any ideas? Thank you!
DpnI will digest the methylated DNA (Your template or Parental plasmid) and what will remain is your amplified mutant plasmid, which is non-methylated, before transformation step so that whatever colonies you will get is from your mutant plasmid and not the parental ones. Most of the common lab bacterial strains are Dam methylation positive which methylate plasmids as well and therefore, for this SDM kit one requirement is to use template plasmid isolated from a Dam positive strain.
DpnI enzyme is specific for methylated and hemimethylated DNA (the plasmid DNA used as templet-DNA in your reaction mixture). So DpnI treatment will digest the templet DNA but it will not affect your newly synthesized DNA containing mutations.
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