QIAprep Spin Miniprep Kit From Qiagen

I have used the QIAprep Miniprep Kit to isolate plasmid DNA for the last five years and feel that it is one of the best kits currently on the market. This kit is extremely easy to use, fast with uncomplicated steps, yields highly reproducible results and plasmid DNA of both high quality and quantity.

The procedure for plasmid isolation consists of three basic steps – preparation and clearing of a bacterial lysate, adsorption of the plasmid DNA on to a silica-gel membrane followed by wash and elution of the DNA.

Upon opening the kit, the first two steps involve addition of RNaseA to the resuspension buffer P1 and addition of ethanol to the wash buffer PE. Once, RNaseA has been added to P1, it has to be stored at 4°C.

The bacteria are first pelleted from an overnight culture in eppendorf tubes. In order to increase the yield of DNA, I typically pellet 1.5 mL bacterial culture twice in the same tube thus obtaining a pellet from 3 mL overnight culture. The supernatant is either vacuumed or pipetted off and resuspended in 250 ìL of buffer P1 using a vortex mixer. The cells are then lysed upon the addition of 250 ìL of buffer P2 and the tube is mixed gently five times by inversion. Then 350 ìL of buffer P3 is added and mixed by inverting the tube 4 to 6 times. The solution becomes cloudy and should not be vortexed or mixed harshly in order to avoid contamination with nuclear DNA. The sample is centrifuged at 13,000rpm for 10 min after which a compact white pellet is seen. The supernatant is applied to a QIAprep spin column by pipetting and the column is then centrifuged for 30 to 60 seconds. The DNA bound to the column is washed once with 0.5 mL of Buffer PB in order to remove trace nuclease activity occurring when using endA+ strains such as JM series or HB101 and its derivatives. This is followed by a wash with 0.75 mL of buffer PE. The flow through is discarded and the centrifugation is repeated in order to remove any residual buffer PE from the column. 50 ìL of Elution buffer is added to the column and let stand for one minute after which the DNA is eluted by centrifugation at 13,000 rpm for 1 min. This DNA can now be used for restriction digestion, sequencing or any other downstream steps. The typical yield of DNA (depending of plasmid copy number etc.) is 5 to 10 ìg DNA from a 3 mL overnight culture.

I have used this kit to purify plasmids ranging in size from 3 kb to 10 kb. The kit worked consistently well in all cases and I have been able to obtain sequencing-quality DNA so far. I have used the purified DNA for subcloning, transfections and transformations.