make a Mega plasmid mutation - (Dec/17/2010 )

Hi there.

I'm study a Mega plasmid;220kb from Environmental strain.

in this case, i want make random mutation or point mutation.

how can i do that?

it's size make me crazy.

some ecoli strains contain lambda phage red recombination system. you can design two homology arms around your mutation site and transform it into this ecoli strain containing your huge plasmid. typically the mutation can be introduced into the plasmid by recombination. in our lab we often use this strain to construct vectors.
however there are many practical problem using this method. you can send me message if you are considering to use this method.