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


High-quality plasmid DNA for all demanding molecular biology applications

Scott Basehore Jeff Braman
Stratagene

A new technique is now available to quickly, easily, and reliably isolate high-purity plasmid DNA. The convenient spin-format eliminates the need for resin suspensions, gravity flow columns, and alcohol precipitations, while easily accommodating simultaneous multiple purifications. The purified DNA is ready for all applications, including sensitive automated sequencing and mammalian cell transfection.

StrataPrep midiprep kit purification of plasmid DNA1 is based on alkaline lysis of bacteria and binding of the liberated plasmid DNA to a glass fiber matrix contained in a midispin cup. Washing the spin cup removes contaminants, and the DNA is eluted in a small volume of buffer. The new midispin cup design and specially formulated alkaline lysis reagents allow up to 350 g of plasmid DNA to be recovered from a single device. The resulting DNA can be used for applications such as restriction digestion, cloning, PCR, site-directed mutagenesis, manual and automated fluorescent sequencing, and mammalian transfection.

Endotoxin, prevalent in plasmid preparations, is difficult to separate from plasmid DNA and results in toxicity to cultured mammalian cells.2 The StrataPrep EF midiprep protocol provides an optional extraction of endotoxins, which are reduced to 0.1 to 0.2 EU/g DNA (endotoxin units per microgram of plasmid DNA); hence, high-efficiency transfection results.

Fast and Easy Method

Fig.1

The StrataPrep midiprep procedure includes a unique color-indicating method that facilitates successful DNA purification (Figure 1). A modified alkaline lysis and centrifugation produces a cleared 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 plasmid DNA was tested in automated fluorescent sequencing. Plasmid DNA purified by a StrataPrep standard midiprep, a StrataPrep endotoxin-free midiprep, twice-banded CsCl density gradient centrifugation, and anion exchange methods were sequenced at Sequetech Corporation. High-signal, low-background sequence data was obtained with all four plasmid preparations. Figure 4 shows representative sequence data for StrataPrep endotoxin-free midiprep purified DNA. Sequence read length for the four templates was approximately 680 bases. There were a number of sequence ambiguities generated from each template, starting arbitrarily from nucleotide 20: one for a StrataPrep midiprep standard, one for a StrataPrep midiprep endotoxin-free, zero for CsCl density gradient centrifugation, and four for anion exchange.

Figure 4

Increased Transfection Efficiency

Endotoxins are toxic to mammalian cell cultures and can greatly affect transfection efficiencies. The StrataPrep midiprep endotoxin-free extraction protocol reduces endotoxin levels to between 0.1 to 0.2 EU/g plasmid DNA. LipoTAXI transfection reagent3,4 and calcium phosphate5 reagent methods were used to transfect StrataPrep endotoxin-free purified DNA into Cos-7, DU 145, and JAR cell lines and resulted in higher transfection efficiencies, compared to anion exchange enndotoxin-free purified plasmid DNA (Figure 5).

Figure 5

Supplemental Components

Endonuclease A removal buffer is available separately when purifying plasmid DNA from End A+ bacterial strains such as JM101. A simple wash step removes endonucleases from the preparation, eliminating non-specific degradation of the plasmid DNA due to the bacterial strain used.

Pre-qualified pyrogen-free 50-ml conical tubes are also available for the preparation of endotoxin-free DNA for sensitive experiments. New plasticware is also considered endotoxin-free.

Conclusions

Use the fast, easy StrataPrep EF plasmid midiprep kit to isolate high-purity endotoxin-free plasmid DNA without resin slurries, toxic chemicals, or specialized equipment. With its convenient format, perform multiple preparations simultaneously, and generate yields similar to maxiprep scale purification. StrataPrep midiprep purified DNA results in accurate automated fluorescent sequence data with high intensity and low background, and the endotoxin-free DNA is transfected into mammalian cells with high efficiency.

REFERENCES
  1. Braman, J. and Basehore, S. (1997) Strategies 10: 84-86.

  2. Cotton, M., et al. (1994) Gene Therapy 1: 239-246.

  3. Pingerelli, P., Petre, M., and Karmiol, S. (1997) Strategies 10: 8-9.

  4. Stratagenes LipoTAXI Transfection Reagent Manual.

  5. Spector, D.L., Goldman, R.D., and Leinwand, L.A. (1998) In Cells: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York. p. 86.2.


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