UltraClean® 6 Minute Mini Plasmid Prep Kit from MoBio Laboratories

Although plasmid purification is routinely performed and requires little skill, the resulting DNA needs to be of high quality in order to obtain the best results in subsequent applications and experiments. Hence, the choice of a plasmid extraction kit is an important one.

The UltraClean® 6 Minute Mini Plasmid Prep kit allows the recovery of high yields of pure plasmid from E.coli strains. In our lab, we usually obtain plasmid DNA that gives an A260/A280 absorbance ratio of 1.8-1.9. The time spent for the isolation is less (6-10 minutes for each miniprep) than competitors, allowing multiple isolations to be performed subsequently (up to four preps without increasing the time).

The protocol describes an effective double centrifuge step to totally remove the culture medium (1 - 2 ml of bacterial culture of starting volume) from the cell pellet; this greatly enhances the DNA yield. Furthermore, another critical step is the resuspension of the cell pellet before lysis. If this is not performed properly then subsequent lysis will be not efficient. The manufacturer suggests two ways for an effective cell resuspension in the appropriate buffer: the first one is by bumping on a vortex for 1 minute (10 seconds on vortex- 1 second out – 10 second on vortex and so on until no cell clump are visible); the second one is to scrape the tube tips back and forth over the holes of an 80 well microcentrifuge tube rack.

Following resuspension, the cell lysis step is carried out in the routine SDS/NaOH cell lysis solution which permits the simultaneous rupturing of membranes, denaturation of chromosomal DNA, and denaturation of plasmid DNA; the 2 strands of the latter remain linked in double circles. At this step, RNAs are also digested by the RNase A enzyme which is added with the resuspension solution.

Then the neutralization step permits the renaturation of plasmid DNA which will reanneal much faster than the chromosomal DNA. Proteins and chromosomal DNA precipitate. After a short centrifuge step, the supernatant is transferred on to a spin filter which binds plasmid DNA in high salt conditions. After a wash step of the spin filter with EtOH/Tris/NaCl, the plasmid DNA is eluted with 10mM Tris and is ready for downstream applications.

The manual supplied in the kit shows the advantages of the kit in comparison with the kit of a direct competitor. There is a complete troubleshooting guide and also an odd section dedicated to the users of the competitor's kit describing the main differences between the kits.