Hi,

I am working with a plasmid which is known to have very low copy number and when I am isolating it by midiprep alkaline lysis method, I am getting good amount of plasmid i.e., 16.56ng/ul. wherein the intensity of the band is good. I have cloned a gene in this plasmid and now when I want to confirm this clone then the my plasmid isolation is showing very low concentrations of plasmid and clone in maxiprep plasmid isolation by alkaline lysis method. Ideally in maxiprep by alkaline lysis method the concentration should be high compared with midiprep by alkaline lysis method. But still the concentration of plasmid and clone is very less which is seen in the gel image of it. It was around 0.80ng/ul for the plasmid and 0.55 ng/ul for the clone. I want to use it for Restriction digestion to confirm the clone wherein I require more concentrations of plasmid DNA and Clone and also require it for sequencing. I am checking each step precisely for troubleshooting. I have checked all the buffers its pH and its storage as well. Solution-2 I am preparing freshly while doing the experiment as mentioned in the protocol. I am not understanding were it has gone wrong.