HiSpeed Plasmid Mega and Giga EF Kits

• Approximately 50 minutes prep time
• Lysate clearing and isopropanol precipitation without centrifugation
• No risk of DNA pellet loss during precipitation
• Up to 10 mg yield of high-copy plasmid DNA
• LyseBlue for optimum lysis and maximum DNA yield
• Endotoxin removal included (<0.04 EU/μg DNA)

HiSpeed Plasmid Mega and Giga EF Kits provide vacuum-driven, large-scale, anion-exchange-based plasmid DNA preparation and require only one centrifugation step to elute the final plasmid DNA. The purified DNA is equivalent to that obtained by 2 x CsCl gradient centrifugation and is suitable for transfection-grade applications. A QIAvac HiSpeed LS vacuum manifold is required for the protocol.

The HiSpeed EF Mega Giga Kits must be used together with the QIAvac HiSpeed LS.

HiSpeed Plasmid Mega and Giga EF Kits contain QIAfilter Cartridges, HiSpeed Tips and QIAconcentrator modules for fast, large-scale plasmid preparation with vacuum manifold. The vacuum driven QIAfilter module replaces the centrifugation step in the classic anion-exchange procedure, making purification faster and more convenient. The QIAconcentrator delivers highly concentrated DNA by centrifugation after ethanol precipitation. Up to 2.5 mg (Mega) or 10 mg (Giga) of high-copy plasmid DNA can be purified from 500 ml or 2.5 l culture, respectively (culture volumes depend on plasmid copy number, size of insert, host strain and culture medium). HiSpeed Tip design allows a very high flow rate, permitting DNA binding, washing and elution steps for plasmid purification to proceed faster. HiSpeed derived plasmid DNA is endotoxin free (<0.04 EU/µg DNA).

The unique anion-exchange resin in HiSpeed Tips is developed exclusively for the purification of nucleic acids. Its exceptional separation properties result in DNA purity equivalent or superior to that obtained by two successive rounds of CsCl gradient centrifugation. HiSpeed Plasmid Mega and Giga EF Kits remove bacterial endotoxins which are released during the lysis step and influence transfection of DNA into primary cells and sensitive cultured cells. The endotoxin-free DNA obtained from the HiSpeed Plasmid Mega/Giga EF Kits is highly suited for reproducible and reliable results in transfection. QIAGEN ultrapure endotoxin-free DNA is also suitable for gene therapy research and other sensitive applications.

QIAfilter Cartridges (see figure “ QIAfilter Mega-Giga Cartridge”) are special filter units designed to replace the centrifugation step following alkaline lysis of bacterial cells. QIAfilter Cartridges completely remove SDS precipitates and clear bacterial lysates in a fraction of the time needed for centrifugation. Prepacked HiSpeed Tips operate by gravity flow and never run dry, minimizing the hands-on time required for plasmid preparation.

The unique QIAconcentrator (see figure “ HighSpeed Plasmid Giga EF procedure”) replaces the centrifugation step traditionally used to collect isopropanol-precipitated DNA following purification. The QIAconcentrator module traps the precipitated DNA, while the isopropanol-buffer mixture flows through. The DNA is then simply eluted from the QIAprecipitator into a collection tube with TE buffer or water. This unique module also eliminates the risk of pellet loss, which can occur during decanting of the supernatant following centrifugation.

Endotoxins, also known as lipopolysaccharides or LPS, are cell-membrane components of Gram-negative bacteria such as E. coli (see figure “ Bacterial cell wall”). Endotoxins are released during the lysis step of plasmid purification and significantly reduce transfection efficiencies in endotoxin sensitive cell lines. Furthermore, endotoxins can influence the uptake of plasmid DNA in transfection experiments by competing with DNA for “free” transfection reagent.

Endotoxins also induce nonspecific activation of immune responses in immune cells such as macrophages and B cells, which can lead to misinterpretation of transfection results. These responses include induced synthesis of proteins and lipids such as IL-1 and prostaglandin. Overall, endotoxins represent a noncontrollable variable in transfection experiment setup, influencing the outcome and reproducibility of results and making them difficult to compare and interpret. In gene therapy research, endotoxins can interfere by causing endotoxic-shock syndrome and activation of the complement cascade. >

Neutralized bacterial lysates are incubated in the QIAfilter Cartridge and cleared in seconds by vacuum. At this stage, the Endotoxin Removal Buffer is added to the filtered lysate. No incubation is needed. The filtrate is applied to a HiSpeed tip for plasmid DNA purification (see figure “ HighSpeed Plasmid Giga EF procedure”). Eluted DNA is mixed with isopropanol and applied to the QIAconcentrator using vacuum and washed. The concentrated and desalted DNA is then eluted from the QIAconcentrator directly into a fresh collection tube with TE buffer or water.

DNA purified with HiSpeed Plasmid Mega and Giga EF Kits yield excellent results in all applications, from cloning and sequencing, to transfection of very sensitive cells and primary cells, and plasmid-mediated gene silencing.