I have some problems with a plasmid construction, I´m using a pET vector (5 kb) where I have already fused my gen of interested and GFP. So, what I´m doing is first digest the plasmid to get rid of the GFP gen, then I do an agarose gel to check if it did cut and I purified the plasmid without the GFP. So then I double digest to insert this genes of aprox 500bp, I purified the digestion of the plasmids and the inserts (I obtained the inserts by PCR and purified them before the digestion) and then I continue doing the ligation, i left it at room temperature overnight. The next day I do the transformation in the competent cells. I do a negative control, but the problem is I´m having colonies in the negative control plate. I think positive and do some minipreps and a restriction to se if I have actually the insert, but no, the gel doesn´t show the insert... just a single scatterd among the line. I decided to run a gel of the minipreps (without digestion) and I´m having bands over the 10 kb band of the ruler, and in others preps It shows two bands aprox 10 kb and 5 kb each one.
So I don´t know from what are those bands and where is exactly my problem.
I need your help, thank you for your time