Improved Alkaline Lysis Method for Rapid Isolation of Plasmid DNA

Isolation of highly purified plasmid DNA from E. coli is an essential step in many techniques of current molecular biology. Traditional techniques for plasmid isolation are the alkaline lysis technique of Birnboim and Doly (2) and the boiling method of Holmes and Quigley (4), which are normally followed by a further purification by cesium chloride gradient centrifugation (9). Though the purity of the plasmids purified by these methods is sufficient for most routine uses, these procedures are both expensive and time-consuming. Many investigators have proposed alternative methods for isolating plasmid DNA on a small or large scale while eliminating the above drawbacks (1,6,10–12), but sometimes these methods are not very timeor cost-efficient. In addition, the partially purified plasmid DNA can only be used for less demanding applications. Recently, we have developed an improved alkaline lysis method for plasmid purification that simultaneously maximizes time efficiency, yield and quality. The procedure uses alkaline lysis, high-concentration potassium acetate precipitation of cellular debris that can eliminate bacterial protein in supernatant efficiently and improve plasmid yield (3), enzymatic digestion of RNA (5) and recovery DNA with PEGMgCl2 (13% polyethylene glycol in 10 mM MgCl2 at room temperature), which gives better recovery and takes less time than the PEG-NaCl method (7). Our protocol consistently provides high-quality plasmid DNA suitable for most routine uses in molecular cloning, and the whole process for a minipreparation can be easily completed in less than one hour with plasmid yields up to 8–9 μg/mL culture for high-copy-number plasmids such as pUC19 and pBluescript (both from Stratagene, La Jolla, CA, USA). The procedure is outlined in Table 1. Our modified protocol does not require toxic reagents such as phenol/ chloroform, and the whole procedure can be carried out at room temperature. In the conventional method, it is always difficult to resuspend the bacterial cells completely with GTE by vortex mixing, but in this procedure, using a toothpick can make it easy. It is important to centrifuge the tubes at room temperature in step 6 because at high salt concentration, low temperature will lead to protein contamination and salt precipitation. Both lowand high-copy-number plasmids isolated with this method are largely supercoiled (Figure 1), and the optical density (OD) ratio at 260/280 nm is 1.92–2.0. These plasmids are suitable for all routine use such as restriction enzyme digestion analyses (Figure 1), double-stranded DNA sequencing (Figure 2), polymerase chain reaction (PCR) etc. Also, this laborand time-saving method has proven to be very helpful in screening hundreds of recombinant clones at one time. It can be easily scaled up for a large-scale preparation of plasmid DNA with its yield up to 6–7 mg DNA/L culture for high-copy-number plasmids. Since the main reagent cost in this method is associated with the use of RNase A, the method represents great reduction in cost compared with the commercial kits (8) and is thus especially attractive for some laboratories where the reagents cost, rather than the labor cost, is the main concern. In conclusion, our improved alkaline lysis method has the best features for a purification method: high yield and purity of the final products, low