Promega’s Wizard® Plus
SV Minipreps DNA Purification System is a simple and reliable method for rapid isolation of plasmid DNA. The procedure yields high quality plasmid DNA in around 30 minutes. Using this kit, 1-10 ml of bacterial culture typically yields up to 25 ug of high quality plasmid DNA depending on a number of factors, including the plasmid copy number, cell density of bacterial culture, type of culture medium and bacterial strain used.
The steps in the given protocol are very easy to follow. When working with high copy plasmids, I recommend strictly following the protocol. Be careful not to use rich media, such as Terrific Broth (TB), which produce high cell densities that may overload the column. Also, old cultures (i.e. cultures grown for greater than 16 hrs) may give low yield of DNA due to cell death and lysis within the culture, so it is also important to ensure that the optical density A600 should remain within 2-4 before harvesting for plasmid isolation. The system also includes the use of alkaline protease which effectively inactivates the endonucleases and other proteins released during the lysis of the bacterial cells than can adversely affect the quality of the isolated DNA. When using alkaline protease in step IIIB, it is also important not to exceed the 5 minute incubation with alkaline protease. I have observed plasmid nicking in such cases.
In principle, the Wizard® Plus SV Miniprep DNA Purification System can be used to isolate any plasmid from E. coli hosts but works most efficiently with plasmids less than 20 kb in size. Plasmids larger than 20 kb are eluted less efficiently from the column and need some extra work. There are 2 steps that I would recommend adopting since plasmids of over 20 kb give a slightly lower yield:
1. In the precipitation step, perform a quick spin to get rid of your proteins and other "rubble"; do not spin too long. The reason for this is that you will also lose part of your plasmid due to its size.
2. When eluting the plasmid off the column in the last step of the protocol, use pre-warmed elution buffer (80ºC). The plasmids bind to the column very strongly (again due to their size) and using warm buffer gives more efficient elution.
The product protocol contains separate protocols for removal of the cleared lysate, i.e. a centrifugation protocol and a vacuum protocol; both are easy to follow. At the end of the protocol, there is a trouble shooting guide that I would recommend to go through as it also helps a lot to understand the potential steps in the procedure that may result in no or low yield of the plasmid DNA.
In conclusion, Promega’s Wizard® Plus SV Miniprep DNA Purification System is very useful and convenient for isolating routinely used plasmids in the laboratory.