Hi all, 

I am trying to make a construct by Gibson Cloning (NEB Kit). I have assembled 4 fragments and the total size is 6.5 kb. These fragments contain some intronic sequences from zebrafish and also GFP. I have followed exactly the same protocol stated on the NEB website to assemble these fragments. I got 10-12 colonies. The competent cells are also from NEB (C3019, highly efficient competent).  I inoculated these colonies in 3 ml ampicilin medium for miniprep but I got very small, hardly any visible pellet. I used home-made reagents. Then I loaded 4.5 ul on the gel (1%). To my surprise, there was a band of thin line width otherwise we normally get a blob on the gel if I load 4 ul. This band was out of the ladder's range. I assume that this could be nicked circular form of the plasmid. I also see one band at 1.25 kb. I am not sure origin of this band or it's a highly supercoiled form of the plasmid. Since I wanted to carry out RE analysis to confirm, I tried to perform midiprep. I inoculated a single colony in 50 ml ampicilin medium and shook for 12-14 hrs. That's how I normally do. I used QIAGEN midiprep kit. Again, I could hardly see any pellet. When I measured the conc., it turned out to be only 17 ng. Still, I loaded some 15 ul out of 20ul on the gel, I do see these two bands but the problem is the yield is too low. I can't use it for any further work. I am re-transforming the remaining plasmid into home-made DH5alpha cells if these commercial cells are having any problem of retaining plasmids in the cells. Another approach I learnt from the research gate is to quadruple the amount of antibiotic. I will also do that. If anyone has any suggestion or faced the same problem, let me know.