Purification of plasmids from bacteria is critical for a variety of molecular biology applications; however, traditional methods for plasmid purification include multiple steps and can be time-consuming. The QuickLyse Miniprep Kit, available from Qiagen, allows rapid purification of plasmids in as little as nine minutes from start to finish, considerably faster than Qiagen’s traditional QIAPrep Miniprep Kit. The kit contains all buffers required for purification as well as spin columns. Prior to use, RNase A and Lysozyme (provided) are added to Buffer QLL to prepare the Complete Lysis Buffer, and isopropanol (not provided) is added to the wash buffer, Buffer QLW.
In this kit, the bacterial pellet resulting from centrifugation is resuspended and lysed in Complete Lysis Buffer by vortexing for 30 seconds, followed by a three minute incubation at room temperature. Bacterial lysates are then applied directly to the Qiagen spin column and centrifuged for 30-60 seconds; the provided wash buffer (Buffer QLW, with isopropanol) is then added directly to the spin column without removal of the flow-through, and centrifuged for 30-60 seconds. The flow-through is then removed, and the spin column is centrifuged again for 60 seconds to remove the remaining isopropanol from the column. The DNA is then eluted from the column using the provided elution buffer (Buffer QLE), and is ready for downstream applications. The rapidity of this miniprep kit compared with other miniprep kits available from this manufacturer results from several innovations: the same buffer is used for both bacterial resuspension and lysis, the lysates are applied directly to the spin column without the need to clear the lysate with a long centrifugation step, and the wash buffer is added to the spin column without removal of flow-through. This kit can also be used for large plasmids if the elution buffer is heated to 70°C prior to elution.
Our laboratory has used the QuickLyse Miniprep Kit to purify plasmids of differing sizes (~ 4.5-12 kb), and, although we enjoy the rapidity of the purification, we have found that the DNA has reduced quality when compared with DNA purified using the standard QIAPrep Spin Miniprep Kit, also available from Qiagen. Based upon discussions with Qiagen technical support personnel, it appears that some of the changes that increase the speed of plasmid purification may also decrease the quality of the eluted DNA. In particular, the absence of a lysate clearing step may cause the spin columns to become overloaded, which can decrease the quality of eluted DNA. Their recommendation was to decrease the size of the bacterial pellet used for the miniprep and perform an additional wash step using a guanidine isopropanol mixture prior to washing with the Buffer QLW wash buffer provided with the kit. However, the addition of an extra wash step increases the time needed for plasmid purification.
In spite of the lower quality of DNA purified using the QuickLyse Miniprep Kit, we have used DNA purified using this method for a variety of downstream applications, including restriction digests, subcloning, PCR, and DNA sequencing, and have found that only DNA sequencing suffers from the reduced DNA quality. As such, the QuickLyse Kit provides DNA suitable for non-sequencing applications.