( Miniprep and midiprep quantities ...)Easy Cosmid DNA Purification with StrataPrep Kits

Miniprep and midiprep quantities of pure high-kb DNA

Jeff Braman Scott Basehore
Stratagene

The StrataPrep plasmid miniprep and midiprep kits are designed to isolate up to 20 and 350 g, respectively, of high-purity plasmid DNA from a single spin cup.^1,2 These kits can also easily purify very large plasmids, including cosmid DNA.

To demonstrate cosmid purification, a SuperCos I clone containing a 40-kb insert from human chromosome 11 was isolated from two, 100-ml overnight cultures using the StrataPrep EF plasmid midiprep kit. The cosmid DNA yield from the two cultures was 45 and 51 g, respectively. The A₂₆₀/A₂₈₀ nm ratios were 1.8 and 1.9, and the A₂₆₀/A₂₄₀ nm ratios were both 1.7, indicating an absence of protein, SDS, and EDTA contaminants.³ Contaminating chromosomal DNA was also absent. The resulting cosmid DNA was digested to completion with EcoR I (Figure 1).

Fig.1

Cosmid DNA can be also be isolated from 1.5-ml overnight cultures using the StrataPrep miniprep kit. The cosmid DNA is completely digested with restriction enzymes such as EcoR I (Figure 2).¹

Fig.2

The midiprep purified cosmid DNA was subjected to automated fluorescent sequencing, a very sensitive application demanding pure template DNA. A representation of the high-quality sequence is shown in Figure 3. The read length was approximately 630 bases with only four sequence ambiguities.

Fig.3'"/>

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