Hi there,

With a picture comparing uncut and cut plasmids, it would be better.

Are you sure of the starting plasmid and of your gel calibration?

I would advise to run double cut and single enzyme cut vector running at same gel. With each single enzyme cut you should see a band of 5308. If you see a band of 3846 bp, it will mean that vector has a problem.

Hi there,

When I took a look at the sides of your bands, I could see  that 3962 bp minus 1346 bp is equal to 2616 bp.

That is quite close to 2500 bp. So I personally think that EcoR1 or Not 1 is cutting your vector more than twice, Leading to two DNA fragments with a length close to 1346 bp for each, and a third fragment close to 2500 bp.

Check your templates for the different EcoR1 and Not1 sites on  your plasmid.