I assembled three fragments using NEB assembly. The colony PCR showed many colonies as positive. I did midi prep to use for subsequent transformation. Now gel, I see the band corresponding to the plasmid (7.1 kb) and an addition band whose size corresponds to the size of my insert (4.7 kb). Why?
Hi there,
Do you run native recombinant plasmid on gel? If yes it's normal to observe several bands as the plasmid exhibits different topological forms (supercoiled, circular relaxed (nicked), linear...). If you want to accurately estimate its size you should digest it at least to linearize it or even to double digest it to estimate its content.
Hi there,
Do you run native recombinant plasmid on gel? If yes it's normal to observe several bands as the plasmid exhibits different topological forms (supercoiled, circular relaxed (nicked), linear...). If you want to accurately estimate its size you should digest it at least to linearize it or even to double digest it to estimate its content.
You can't accurately judge plasmid size unless you digest it first. Choose an enzyme that will cut your plasmid in ONE spot, make sure it does not cut your insert, and run out your linearized plasmid on a gel. As Dominique said, you are most likely seeing the different forms of just your plasmid. Good luck!
I used both single and double digestion and see the bands I expect. Thanks a lot.
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