The existence of mixed plasmids in the same cell is tricky and there is a need for separating them from each other. However, isolating two existed plasmids might be difficult, particularly if they are same in their sizes, with same antibiotic marker, or different only in one or more mutants without different restriction cut. Two different plasmids in the same cells is a natural phenomenon as well as a normal practice in molecular biology experiments. For example during random mutagenesis experiment for a single gene (existed naturally in an operon) using a mutator strain like E. coli XL1 Red, the single mutated gene is then complemented with the other essential genes for producing certain products. Another example, during site directed mutagenesis experiment using double antibiotics selection method, in many cases, the original plasmid is existed side by side with the one carry the new mutant. There are many examples where plasmids coexisted with each other either naturally or under experimental conditions. The problem is how one could separate those plasmids particularly when they are similar in their molecular weight and have the same marker. This study introduces two main strategies; the first is based on increasing or decreasing the competent cells transformation efficacy. Where, in general harvesting competent cells either E. coli or other bacterial strains in the first 2-3 hours (or less) of their cultivation and using the enhanced protocol for competent cells preparation will improve the transformation processes. Letting cells to be more ages will reduce the transformation processes. Using four 2-3 hour grown competent Azotobacter sp enable plasmid transformation. The second strategy for separating the coexisted plasmid is based on using diluted plasmids. The antibiotic screening method is based on blind selection where growing on plat containing the first antibiotic and non growing in the second antibiotic means that the tow existed plasmids are separated. In case of existing of plasmids with the same size and the same antibiotic marker for example during the site-directed-mutagenesis protocol (mutants did not have different restriction enzymes cut), the plasmid is diluted and transformed in recombinant E. coli and each clone was cultivated alone and the mutated region is sequenced. The presence of a single base pair in the site of the mutant means presence of a single plasmids and vice versa. As a conclusion same plasmids with point mutation are usually coexisted. In some cases the coexisted plasmids are with similar antibiotic marker, no different restriction enzyme cut sites are existed, no white and blue selection or any other phenotype for selection. In such cases and similar ones diluting plasmid and transforming them in conditions enable single plasmid per cell must be controlled by the sequencer. The protocols included in this study are summarized from the experiences with random and site directed mutagenesis experiments where plasmid with a single mutant is coexisted with the wild mother plasmid or with the other coexisted different mutants.
A mutant screening method by critical annealing temperature-PCR for site-directed mutagenesis