I am doing some standard ligation cloning here and running into a systematic issue.
I am attempting to ligate a 1 kb insert (obtained by overlap extension PCR) into NcoI and KpnI sites of a particular plasmid. I get plenty of colonies on my transformations, and very few colonies when the insert is left out of the ligation.
I sequenced 4 colonies so far, using two different forward primers and one reverse primer for each colony. All sequencing primers lay down on the plasmid backbone just before (and after in the case of the reverse primer) where I would expect the insert to be, and should read into the insert.
The reverse sequencing comes back exactly as expected. The insert is there and all the mutations I have made that can be seen from the reverse direction are present.
However, every single forward sequencing reaction comes back with overlap traces, irrespective of the sequencing primer used. The overlaps in the sequencing traces start about 10 bases upstream of the NcoI site, i.e. just before the insert.
The only thing that I can think of is that somehow the plasmid is ligating to another plasmid molecule through their NcoI sites? This would create two sites for the forward sequencing (but also two sites for reverse sequencing...which doesn't make sense).
Cloning is not a new thing for me but this particular issue is, and any suggestions as to the source of the sequencing overlaps would be most appreciated.
I am redoing the ligation using the same plasmid backbone from a different source in case the plasmid itself that I used just happened to be bad.