Principle

Time and cost efficient high-throughput purification of plasmid DNA

The NucleoSpin® 96 Flash kit is designed for the rapid, high-throughput isolation of high copy plasmid, cosmid, and BAC DNA using vacuum manifolds or centrifuges. The final precipitation requires the use of a centrifuge. The kit allows fast and reliable purification of up to 8 μg of plasmid DNA per preparation from 1.1–1.3 mL of a saturated E. coli culture and ensures reproducible yields of DNA in the convenient 96-well format. Cultivation of E. coli in rich media like LB, 2xYT, or TB is recommended. For BACs, yields of 0.1–0.2 μg/mL culture can be achieved. With the supplied culture plate E. coli cultures can be grown in a volume of up to 1.5 mL. Processing of E. coli cultures grown in up to 5 mL LB, 3 mL 2YT, or 3 mL TB is possible when using suitable culture tubes or plates (not supplied with the kit).

The plasmid DNA purified with NucleoSpin® 96 Flash is suitable for e.g. routine sequencing, PCR, enzymatic reactions, and screening applications. With the NucleoSpin® 96 Flash method plasmid DNA is liberated from the E. coli host cells by SDS/alkaline lysis after resuspension of the bacteria pellets. The resulting lysate is then neutralized and cell debris is precipitated together with SDS. Optional incubation in boiling water inactivates nucleases which are present in a variety of E. coli host strains and denatures soluble proteins and other cell constituents. All precipitates are then removed by filtration of the lysate through the NucleoSpin® Flash Filter Plate. Plasmid DNA is collected by addition of isopropanol to the filtrate and subsequent centrifugation. After an additional washing step with 70% ethanol and drying the DNA can be resuspended in 5 mM Tris/HCl, pH 8.5.

Features

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