restriction enzyme digestion - (Jan/04/2009 )
I'm attemping TA cloning using TOPO vector. I only had a single colony on my plate following transformation. After plasmid purification and retriction enzyme digestion, i get a 50bp band and a vector band on my gel. I'm cloning a 1053bp fragment using kpnI and XhoI but I'm getting a 50bp band on the gel?
I added reatriction sites to my primers and I got a 1053bp band o my gel foloing PCR. I had puified this and attempted cloning but I don;t understand the 50pn band? am'I experiencing star activity?
I haven't worked with TOPO vectors but I think what you are getting is religation of vector and the difference between Kpn I and Xho I in the vector would be 50bp, which you see on the gel.
I hope its not RNA which you are misinterpretting as a band?