I want to create a recombinant plasmid with the insert of size 1128 bp and the plasmid pET-15b of size 5708 bp. DNA insert and plasmid vector was double digested with BamH1 and BIpI restriction enzymes. I have done ligation reactions in 3:1, 5:1 and 7:1 DNA insert:vector ratios. Attached is the agarose gel image with the following description:
LANE 1 -1 kb DNA ladder
LANE 3 - Double digested pET-15b plasmid
LANE 4 - Double digested DNA insert.
LANE 6 - Ligated product of 3:1 ratio
LANE 7 - Ligated product of 5:1 ratio
LANE 8 - Ligated product of 7:1 ratio
IMO bands in lanes 6,7,8 that are above a band of lane 3 can be interpreted as products of ligation; i.e., circular plasmids. Circular plasmids tend to have lower mobility.
When such analysis is done it is crucial to have a negative control. As a negative control two reactions can be set up. First, a reaction with backbone and insert, but no ligase. Second, a reaction with backbone and ligase, but no insert.
It is hard to know if 6, 7, 8 are the plasmids you want or just some background. You should run those samples digested with the same cloning enzymes to be certain.
So it is true that you obtained circular plasmid from your minipreps but you don't actually know what they are. They could be contaminants, could be only the vector, could be an incorrect ligation product.
You need to at least confirm that the vector band and the insert band are the correct size before proceeding.
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