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A Simple and Rapid Technique to Purify High-Quality Midiprep DNA

eared bacterial lysate. A chaotropic salt is added to the lysate, and the mixture is transferred to a midispin cup. Upon centrifugation, plasmid DNA binds to the glass fiber matrix in the midispin cup. Contaminants are removed by washing the midispin cup, and the plasmid DNA is eluted in a small volume of buffer. No alcohol precipitation is necessary to concentrate the DNA. Endotoxin is efficiently removed by optionally extracting the cleared bacterial lysate prior to adding the chaotropic salt. In one hour, the standard midiprep plasmid purification is complete (only 1.5 hours with endotoxin removal); the standard anion exchange method takes a minimum of 3 hours to complete.

High Yields and Purity

Fig.2

The maximum binding capacity of a StrataPrep midispin cup is 350 g of plasmid DNA (Figure 2). The binding capacity of a single StrataPrep midispin cup was determined by loading various known quantities of purified pBluescript plasmid into midispin cups, then washing and eluting the DNA. The quantity of eluted DNA was measured by UV spectrophotometry. Nearly 350 g of high-copy number pBluescript II SK(+) plasmid DNA was also recovered from a 100-ml overnight bacterial culture using a single midispin cup, while the low-copy number pCMV gal plasmid yield from a 200-ml overnight culture was 250 g (Figure 3). Yields and spectrophotometric values (A260/A280 nm) between 1.8 and 2.0 were identical, using both the standard and endotoxin-free protocol (data not shown).

Figure 3

Quality Automated Sequencing

The quality of StrataPrep midiprep plasmi
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