Problem with plasmid linearization - (Jun/29/2004 )

Hi all,

I have done a restriction digest for plasmid linearization and subsequent yeast transformation.

The problem is that after digestion the plasmid migrates in two very similar sizes, both of them higher than those of unlinearized plasmid.

A linearized plasmid migrates in an unique size, doesn't it?

Thanks a lot for all suggestions

Hi there!
A linearised plasmid should run at the size corresponding to its actual bp length. Uncut or nicked plasmids may run at various sizes, but should be consistent from time to time. Your 2 bands could be uncut and nicked DNA, ie some partial digestion maybe?

Hi!

I agree with the above answer. I did have some problems with digestion similar to what you are discribing. How much template (plasmid) are you using in your digestion rxn? You might try using less of your template.