Comparing Midiprep Systems
We compared PureYield™ Plasmid Midiprep System to the following plasmid midiprep systems: CompactPrep® Plasmid Midi Kit, QIAfilter Plasmid Midi Kit, Qiagen® Plasmid Midi Kit and HiSpeed® Plasmid Midi Kit (all Qiagen) in terms of speed, yield, purity and performance of DNA in downstream applications. DNA quality was evaluated by absorbance (230nm, 260nm, 280nm) and by transfection capability.
Of the five kits tested, the PureYield™ Midiprep System gave the highest overall yield of plasmid (Figure 3). The plasmid DNA isolated with all the kits was of high quality according to the A260/A280 and A260/A230 ratios. In terms of speed, the PureYield™ and CompactPrep® kits were quickest (Table 1). Although the fast lysate-clearing step and lack of a need for high-speed centrifugation were advantages of the CompactPrep® kit, the kit can only process 25ml of culture. By comparison, the PureYield™ Midiprep System can process up to 100ml of culture. The PureYield™ System resulted in larger DNA yields than CompactPrep®; the CompactPrep® and HiSpeed® kits gave the lowest overall yields.
Figure 3. Yield and purity measurements of plasmid DNA isolated from PureYield™ and Qiagen® midiprep kits. Cultures of pFC8A-luc with an OD600 of 2.8–3.0 were split into 25ml aliquots. Data are the average of triplicate or quadruplicate (CompactPrep®, PureYield™ and HiSpeed®) 25ml preps. Average yield was calculated from absorbance at 260nm. Average purity was calculated by absorbance at 260nm/280nm and 260nm/230nm.
In addition, luciferase activity from plasmid DNA isolated using the PureYield™ Midiprep Kit and used in transfection exceeded that from plasmid DNA isolated from the Qiagen midiprep kits (Figure 4). These results further demonstrate that the high yield and high quality of plasmid DNA isolated using PureYield™ Midiprep Kit leads to enhanced performance in sensitive downstream applications.
Figure 4. Average firefly luciferase activity in cells transfected with midiprep plasmid DNA. CHO cells (10,000 cells/well) were transfected in a 96-well plate with 0.2µg/well of plasmid DNA. TransFast™ Transfection Reagent was used at 1:1 lipid:DNA ratio in the absence of serum followed by assaying for luciferase activity using ONE-Glo™ Luciferase Assay System. All samples were performed in triplicate.
Comparing Maxiprep Systems
The PureYield™ Plasmid Maxiprep System was compared to the following plasmid isolation kits: HiSpeed® Plasmid Maxi Kit, QIAfilter Plasmid Maxi Kit, CompactPrep® Plasmid Maxi Kit, Qiagen® Plasmid Maxi Kit, and EndoFree® Plasmid Maxi Kit (all from Qiagen). We compared these kits in terms of speed, yield, purity and performance in downstream applications. DNA quality was evaluated by absorbance (230nm, 260nm, 280nm) and by transfection capability.
Of the six kits tested, PureYield™ System again gave the highest overall yield of plasmid DNA (Figure 5). The plasmid DNA isolated with PureYield™, CompactPrep® and Qiagen® Plasmid Maxi kits was of high quality according to A260/A280 and A260/A230 ratios calculated, whereas QIAfilter, HiSpeed®, and EndoFree® suffered from low A260/A230 ratios.
Figure 5. Yield and purity measurements of plasmid DNA isolated from PureYield™ and Qiagen maxiprep kits. pGEM®-3Z Vector was grown in JM109 cells to an OD600 of 4.79. Three hundred microliters of culture was centrifuged at 6,000 x g, and the pelleted material was frozen. Frozen pellets were reconstituted with the appropriate kit buffer and then split into triplicate samples. All kits tested used one-third of each bacterial pellet made from the 300ml culture (or the equivalent of 100ml of culture). Triplicate plasmid preps were performed with each kit. Average yield was calculated from absorbance at 260nm. Average purity was calculated by absorbance at 260nm/280nm and 260nm/230nm.
In terms of time, the PureYield™ and CompactPrep® kits again were the quickest (Table 1). CompactPrep® advantages included no need for high-speed centrifugation steps (aside from a 1-minute elution spin on a benchtop microcentrifuge) and the inclusion of LyseBlue® dye to confirm full neutralization of the lysed sample. But as was the case with the midipreps, in terms of yield, the PureYield™ System held an advantage over CompactPrep®, with greater than a twofold increase in total plasmid yield. The HiSpeed® protocol was significantly faster but at the expense of DNA purity. Finally, long centrifuge and wash times resulted in significantly longer protocols, from 2.5 to >3 hours for the EndoFree®, Qiagen® Plasmid Maxi, and QIAfilter maxiprep kits.
Maxiprep DNA quality was also evaluated by transfection of mouse 3T3 cells with luciferase-expressing pGL3-control plasmid DNA followed by assaying for luciferase activity using the ONE-Glo™ System (Cat.# E6110; Figure 6). The resulting luciferase activity was similar using plasmid DNA from each of the maxiprep kits.
Figure 6. Average firefly luciferase activity in cells transfected with maxiprep plasmid DNA. Mouse 3T3 cells (10,000 cells/well) were transfected in a 96-well plate with 0.2µg/well of pGL3-Control plasmid DNA. TransFast™ Transfection Reagent was used at a 1:1 lipid:DNA ratio in the absence of serum; then luciferase activity was assayed using the ONE-Glo™ System. All samples were performed in triplicate.