HiSpeed Plasmid Purification Midi Kit From Qiagen

The HiSpeed Plasmid Purification Kit fits very well into Qiagen’s established line of products for DNA preparation and purification. The buffers used with this kit are identical to the ones used in a number of other kits from this company, but this kit is really fast and comfortable to use since only one centrifugation step (to harvest the bacteria) is required during the purification. The kit is based on a modified alkaline lysis procedure followed by binding the DNA to an anion-exchange resin under low salt and pH conditions. What differentiates this kit from others is that instead of spinning at high speed, the bacterial lysate can be cleared just by pushing the solution through a QIAfilter cartridge (a filter in a syringe format). Another time-saving tool are the QIAprecipitator modules. In these modules, the precipitated DNA is trapped as a thin layer allowing washing and drying simply by pushing ethanol or air, respectively, through the precipitator module using a syringe.

The whole procedure works as follows: For low copy plasmids, a 150 ml culture is grown overnight (for high copy plasmids a 50 ml culture is recommended). The company also recommends the use of LB medium with 10 g NaCl per liter to get the highest yields of DNA. The bacteria are harvested by centrifugation (6000 x g, 10 minutes, 4°C) and the pellet is resuspended in 6 ml of ice cold Buffer P1. Then 6 ml of buffer P2 (room temperature) is added to lyse the cells. The solution has to be mixed very well to achieve complete lysis and is then incubated for 5 minutes at room temperature. For neutralization of this solution, 6 ml of pre-chilled buffer P3 is added and the solution is mixed immediately. The lysate is then poured into the barrel of the QIAfilter cartridge and incubated at room temperature for 10 minutes without insertion of the plunger into the syringe. The kit includes blue adaptors; with these adaptors, the syringes, cartridges and columns fit nicely in standard 50 ml disposable plastic tubes.

Before you use the QIAfilter cartridge, you have to close the outlet nozzle of the syringe with a cap, which is included in the kit. After the 10 minute incubation, the cap is removed and the lysate is pushed with the plunger through the QIAfilter cartridge. During the 10 minute incubation, one should prepare the HiSpeed Midi column by applying 4 ml of the equilibration buffer QBT and allowing the column to empty by gravity flow. Then one has to apply the cleared lysate from above onto the column and let it drip through by gravity flow. For the washing step, 20 ml of the wash buffer QC sre applied and afterwards the DNA is eluted by adding 5 ml of the elution buffer QF. The DNA is then precipitated by adding 3.5 ml isopropanol to the eluate. During this incubation, the QIAprecipitator Midi module has to be prepared by putting a 20 ml syringe from which the plunger has been removed onto the outlet nozzle. Next one has to pour the DNA precipitate into the 20 ml syringe of the QIAprecipitator module. The plunger is inserted into the syringe and the eluate/isopropanol mixture (DNA precipitate) is pushed slowly through the module. Next, the syringe is removed from the precipitator module and the plunger is taken out. It is important that you always detach the syringe from the precipitator module before you remove the plunger. The syringe is again attached to the module and 2 ml 70% ethanol are added; the plunger is inserted and the solution is pushed through slowly. This will remove any traces of isopropanol from the bound DNA. Air is then pushed through the precipitator module. The outlet nozzle of the precipitator is then dried with a paper towel and the DNA is eluted by pushing 1 ml of TE buffer through the preciptator module using a new 5 ml syringe; the eluate is collected in a 1.5 ml reaction tube. To increase the yield, a second elution can be performed by applying the 1 ml eluate to the syringe again and pushing it through the precipitator module. DNA yield is determined as usual by UV spectrophotometry at 260 nm and/or by analysis of an aliquot on an agarose gel.

This method is really fast and gives good DNA yields. You should sort out all the different steps and look at the contents of the kit before you start, even when you read the manual carefully. It can be confusing the first time with all the steps, which are so similar to the usual purifications using these type of columns, but then there are important additions.