Miniprep and midiprep quantities of pure high-kb DNA
Jeff Braman Scott Basehore
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).
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
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.
The StrataPrep plasmid miniprep and midiprep kits both produce high yields of all sizes of DNA from 2.9-kb plasmids to 45-kb cosmids. Both have rapid protocols using a modified alkaline lysis method to yield quality sequence with automated or manual methods. The amount of purified DNA required determines which kit to use. StrataPrep purified cosmid DNA can serve as the source material for subclone library construction used in shotgun sequencing projects.4