How to extract plasmid DNA from transformants?
Plasmid DNA was extracted from the transformants using the Machery-Nagel NucleoSpin Plasmid kit and digested with EcoRI restriction enzyme. Plasmids were visualized on a 1% agarose gel stained with GelRed and run at 70 V for 60 mins.
Are commercial plasmid extraction kits capable of dealing with complex samples?
It thus appears that these kits were not capable of dealing with the complexities associated with our samples. The alkaline lysis method (Birnboim and Doly, 1979) is a common method of plasmid extraction, on which most commercial plasmid extraction kits are based.
Can we extract plasmid DNA containing antibiotic resistance genes?
The direct extraction of plasmid DNA containing antibiotic resistance genes from complex samples is imperative when studying plasmid-mediated antibiotic resistance from a One Health perspective, in order to obtain a wide representation of all the resistance plasmids present in these microbial communities.
How to purify plasmid DNA from bacterial DNA?
Purification of plasmid DNA from bacterial DNA using alkaline lysis is based on the differential denaturation of plasmid and chromosomal DNA. Disruption of the cellular structure to produce a lysate, separation of the plasmid from the chromosomal DNA, cell debris, and other insoluble material are the essential steps of plasmid isolation.
Are there any plasmid DNA extraction kits for complex samples?
At present, there are no commercial kits designed to extract plasmid DNA directly from complex samples. Current plasmid extraction kits are intended to work with pure bacterial culture, which is less than ideal when dealing with complex environmental samples.
Can plasmid DNA be used for electrophoresis?
Plasmid DNA that has been nicked (covalently opened) will run slower than supercoiled DNA during electrophoresis. A small amount of this species of DNA is common and is suitable for downstream applications. Permanently denatured DNA will migrate ahead of the supercoiled DNA and may not be suitable for downstream applications.